Catalog 2013â2014 Advancing Research with ... - Biocenter
Catalog 2013â2014 Advancing Research with ... - Biocenter
Catalog 2013â2014 Advancing Research with ... - Biocenter
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Bio<strong>Research</strong><strong>Catalog</strong> 2013–2014<strong>Advancing</strong> <strong>Research</strong> <strong>with</strong>Biologically Relevant Solutions––Primary Cells and Media––Media, Reagents and Sera––Mycoplasma Detection andPrevention––Transfection––BioAssay Products and Services––Electrophoresis and Analysis––Services––QC Testing Solutions
Lonza – <strong>Advancing</strong> <strong>Research</strong> <strong>with</strong> Biologically Relevant SolutionsBiological RelevanceOur product offering centers around the use of primary cells and related products to provide biologically relevant solutions foryour research. When performing in vitro research, you need to replicate the in vivo environment as closely as possible. <strong>Research</strong>has shown that primary cells, which are non-transformed, non-immortalized cells isolated directly from tissue, provideconditions that closely simulate a living model and yield more physiologically significant results. Building on that standard, westrive to ensure that all of our products improve the biological relevance of your research results.Integrated <strong>Research</strong> SolutionsWe provide complete and functionally qualified solutions for your cell and molecular biology research applications to improveand accelerate experimental results. For example, you can successfully transfect our Clonetics Primary Cells <strong>with</strong> your gene ofinterest using Nucleofector Devices and Kits and confirm cell viability after transfection <strong>with</strong> the Vialight Bioassay.With our well-known and trusted leading brands for products, we continue to set the standard for quality in the industry.Integrated cell culture solutions span our product areas <strong>with</strong> guaranteed performance. Products include:■■Clonetics Primary Cells and Media – Over 150 authenticated primary cell types and optimized media, ready to use today.■■Stem Cells and Media – Primary adult stem and progenitor cells available <strong>with</strong> growth and differentiation media, andES-derived differentiated cells and media.■■Nucleofector Technology – Proven over the past 10 years as the optimal transfection technology for hard-to-transfectprimary cells and cell lines.■■MycoAlert Assay and MycoZap Antibiotics – Complete portfolio for easy and quick mycoplasma detection, eliminationand prevention.■■BioWhittaker Media and Sera – Classical media, plus an extensive array of serum-free media including protein-free,chemically defined and non-animal derived formulas.■■SeaKem®, NuSieve and MetaPhor Agarose, and FlashGel Systemfor fast reliable separation and analysis of nucleic acidsand proteins.QC Testing SolutionsLonza is the trusted QC testing solution provider for the pharmaceutical and medical device industries. We transform ourpractical knowledge and technical expertise to deliver a portfolio of endotoxin, mycoplasma and microbial detection products,software and services that support the critical needs of regulated manufacturing environments, like our own.Contacting LonzaPurchase any of our products directly, using the contact information provided on page 12 of this catalog, or use our e-commercecapabilities at www.lonza.com. For technical information, please contact our Scientific Support Team. Contact informationprovided on page 13 of this catalog.Based on over 40 years of experience and innovation, we are committed to providing the highest quality products, services andscientific support to advance life science research <strong>with</strong> biologically relevant solutions. For all of your research and QC testingproduct and service needs, turn to us. We appreciate your continued support and look forward to serving you.Your Lonza Bio<strong>Research</strong> Team
About LonzaLonza is a global company serving the life-science industry.Over a century ago, we began as a small Swiss electricitycompany, making a few chemicals on the banks of the riverLonza in the Valais region of the Swiss Alps. More than110 years later, we are a leading supplier to thepharmaceutical, healthcare, and life-science industries.We offer over 4,000 products and services to more than60,000 customers worldwide. Our customers range fromprofessionals <strong>with</strong>in the pharmaceutical, biotechnology,academic, and government research industries tomanufacturers of consumer and health products,distributors, formulators, and service companies.Since 1897, we have used our enterprising character toadapt our offerings and services to your needs and to thechanging technologies.In Pharma&Biotech we partner <strong>with</strong> pharmaceutical andbiopharmaceutical customers for their manufacturingneeds. Using a variety of technologies, we make theingredients used in many critical drugs, treating patients inareas such as cardiovascular diseases, cancer, neurologicaland infectious diseases. Cell therapy companies rely on ourcustom manufacturing services. And our portfolio ofendotoxin, mycoplasma and microbial detection products,software and services supports the critical needs ofregulated manufacturing environments.For Bio<strong>Research</strong> we make the tools you use to develop andtest therapeutics, from basic research through to finalproduct release. These tools range from cell culture andmolecular biology products for life-science research tomedia used in the production of therapeutics and tests formicrobial detection. We offer over 2,000 research productsand services, including human and animal primary cells,cell culture media and sera, transfection technologies,bioassays, as well as electrophoresis instruments andreagents.Other markets we serve are Nutrition, Agriculture,Personal Care, Microbial Control and Materials Science.We have a global network of sales and support offices, <strong>with</strong>representatives who are close to you, speak your languageand understand your needs. Our production and R&Dactivities at 45 sites around the world ensure the closeconnection necessary to best serve your local needs.Primary Cells and MediaMedia, Reagents and SeraMycoplasma Detection and PreventionTransfectionBioAssay Products and ServicesElectrophoresis and AnalysisServicesQC Testing SolutionsTechnical Information1234567893
Table of ContentsHow to Use this <strong>Catalog</strong> 10How to Order 11Terms and Conditions 12How to Get Scientific Support 13Trademarks and Patents 141 Primary Cells and MediaQuick Reference Guide 17Clonetics Human Primary Cells and MediaIntroduction28Bladder Cells and Media 29Cardiac Cells and Media 31Dermal Cells and Media 34Large Vessel Endothelial Cells and Media 37Microvascular Endothelial Cells and Media 39Gastrointestinal Cells and Media 42Lymphatic Cells and Media 43Mammary Epithelial Cells and Media 44Neural Cells and Media 45Ocular Cells and Media 46Pancreatic Islets 47Prostate Cells and Media 48Pulmonary Cells and Media 50Renal Cells and Media 54Reproductive Cells and Media 56Skeletal and Connective Tissue Cells and Media 58Skeletal Muscle Cells and Media 60Clonetics Animal Primary Cells and MediaIntroduction64Cardiac Cells and Media 65Fibroblasts Cells and Media 67Neural Cells and Media 68Ocular Cells and Media 71Skeletal Cells and Media 72Cell Culture Reagents 73Human Neural Progenitor Cells and Media 82Human Osteoclast Precursor Cells and Media 83Human Pluripotent Stem Cells 84Human Preadipocyte Cells and Media 86Human Mesenchymal Stem Cells and Media 88Pluripotent Stem Cell Services 90Rat Mesenchymal Stem Cells and Media 91Poietics Immune Cells and MediaIntroduction93Human Dendritic Cells and Media 94Human CD14 + Monocytes and Media 95Human Natural Killer Cells 96Human T Cells and Media 962 Media, Reagents and SeraBioWhittaker Classical MediaIntroduction100Basal Medium Eagle 102Dulbecco’s Modified Eagle’s Medium 102DMEM:F12 Medium 104Glasgow Minimum Essential Medium 104Grace’s Insect Medium 105Ham’s Medium 105Iscove’s Modified Dulbecco’s Medium 106L-15 Medium 106McCoy’s 5A Medium 107Minimum Essential Medium – Eagle 107Medium 199 109NCTC-109 Medium 109Richter’s CM 109RPMI 1640 Medium 110Insect Medium 111Waymouth’s Medium 111William’s Medium 111Poietics Stem Cells and MediaIntroduction76Human Adipose-derived Stem Cells 77Human Bone Marrow Products 78Quadratox 4 Species Panel of Bone MarrowMononuclear Cells 78Mononuclear Cells 79Human CD34 + and CD133 + Progenitor Cells 80Human CD34 + Differentiation Supplements 814North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Table of ContentsBioWhittaker Specialty MediaIntroduction114TheraPEAK MSCGM-CD Mesenchymal StemCell Growth Medium – Chemically-Defined 115FGM-CD Fibroblast Growth Medium –Chemically Defined 116UltraCULTURE Serum-free Medium 117PC-1 Chemically Defined, Serum-free Medium –Chemically Defined 118UltraMEM Reduced Serum Media 119X-VIVO Serum-free Hematopoietic Cell Media –Chemically Defined 120UltraCHO Serum-free CHO Cell Media 121ProCHO Protein-free CHO Media 122PowerCHO Serum-free CHO Media –Chemically Defined 123ProFreeze-CDM, NAO Freeze Medium –Chemically Defined 124UltraMDCK Serum-free Renal Cell Medium –Chemically Defined 124Pro293 Serum-free Media – Chemically Defined 125ProVero 1 Serum-free Medium 125ProPer 1 Serum-free Medium – Chemically Defined 126PERMEXCIS® Virus Production Medium –Chemically Defined 127Lymphochrome Medium 127Amniochrome II Modified Medium 128Amniochrome Plus Medium 128Amniochrome Pro Medium 128HL-1 Serum-free Media – Chemically Defined 129UltraDOMA Serum-free Hybridoma Medium 131ProDoma Serum-free Hybridoma Media –Chemically Defined 132UltraDOMA-PF Protein-free Hybridoma Media 132Insect-XPRESS Protein-free Insect Cell Medium 133ProNS0 Protein-free Media – Chemically Defined 134BioWhittaker Cell Culture ReagentsIntroduction136Balanced Salt Solutions 137Earle’s BSS 137Hanks’ BSS 137Reagents138Growth Factors 140Antibiotics and Antimycotics 140Penicillin-Streptomycin Mixtures 141Buffers and Buffered Salines 142Viral Serology 144BioWhittaker Animal and Human SeraIntroduction146Fetal Bovine Sera 149Bovine Sera 149Human Sera 150Fetal Bovine Serum 150Equine Sera 1513 Mycoplasma Detection and PreventionIntroduction155DetectionMycoAlert PLUS Mycoplasma Detection Kit –For <strong>Research</strong> Use 157MycoTOOL® PCR Mycoplasma Detection Kit –For Final Release Testing 158Elimination and PreventionMycoZap Mycoplasma Elimination Reagent 160MycoZap Antibiotics 161MycoZap Spray 1624 TransfectionNucleofector TechnologyIntroduction166Components of the Nucleofector Technology 168Advanced Platform: 4D-Nucleofector System 169Adherent Nucleofection 170Nucleofector Devices and Systems4D-Nucleofector System 17296-well Shuttle System 173Nucleofector 2b Device 174384-well Nucleofector System 175Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com5
Table of ContentsNucleofector KitsNucleofector Kits for Primary Cells – OverviewPrimary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 178Adherent Nucleofector Kits for4D-Nucleofector System 182Primary Cell Optimization Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 183Primary Cell Kits for Nucleofector II /2b Device 184Nucleofector Kits for Primary AdipocytesNucleofector Kits for Human Pre-Adipocytes 186Nucleofector Kits for Primary Blood CellsNucleofector Kits for Human B Cells 187Nucleofector Kits for Stimulated Mouse B Cells 188Nucleofector Kits for Human Dendritic Cells 189Nucleofector Kits for Mouse Dendritic Cells 190Nucleofector Kits for Human Macrophages 191Nucleofector Kits for Mouse Macrophages 192Nucleofector Kits for Human Monocytes 193Nucleofector Kits for Human Natural Killer Cells 194Nucleofector Kits for Human T Cells 195Nucleofector Kits for Mouse T cells 196Nucleofector Kits for Mammalian Blood Cells 197Nucleofector Kits for Primary Bone /Cartilage CellsNucleofector Kits for Human Chondroycytes 198Nucleofector Kits for Primary Cardiac CellsNucleofector Kits for Rat Cardiomyocytes 199Nucleofector Kits for Primary Dermal CellsNucleofector Kits for Human Keratinocytes 200Nucleofector Kits for Human Melanocytes 201Nucleofector Kits for Primary Endothelial CellsNucleofector Kits for Human Coronary ArteryEndothelial Cells 202Nucleofector Kits for Human MicrovascularEndothelial Cells – Lung 203Nucleofector Kits for Human Umbilical VeinEndothelial Cells 204Nucleofector Kits for Mammalian Endothelial Cells 205Nucleofector Kits for Primary Epithelial CellsNucleofector Kits for Human BronchialEpithelial Cells 206Nucleofector Kits for Human MammaryEpithelial Cells 207Nucleofector Kits for Mammalian Epithelial Cells 208Nucleofector Kits for FibroblastsNucleofector Kits for Human Dermal Fibroblasts 209Nucleofector Kits for Mouse Embryonic Fibroblasts 210Nucleofector Kits for Mammalian Fibroblasts 211Nucleofector Kits for Primary HepatocytesNucleofector Kits for Human Hepatocytes 212Nucleofector Kits for Mouse or Rat Hepatocytes 213Nucleofector Kits for Primary Muscle CellsNucleofector Kits for Human Aortic SmoothMuscle Cells 214Nucleofector Kits for Human SkeletalMuscle Myoblasts 215Nucleofector Kits for Mammalian Smooth Muscle Cells216Nucleofector Kits for Primary Neural CellsNucleofector Kits for Chicken Neurons 217Nucleofector Kits for Mouse Neurons 218Nucleofector Kits for Rat Neurons 219Nucleofector Kits for Mammalian Neurons 220Nucleofector Kits for Mammalian Glial Cells 221Nucleofector Kits for Primary Stem CellsNucleofector Kits for Human CD34 + Cells 222Nucleofector Kits for Human H9 Stem Cells 223Nucleofector Kits for Human MesenchymalStem Cells 224Nucleofector Kits for Human Stem Cells 225Nucleofector Kits for Mouse Embryonic Stem Cells226Nucleofector Kits for Mouse Neural Stem Cells 227Nucleofector Kits for Rat Neural Stem Cells 228Nucleofector Kits for Animal Stem Cells 229Nucleofector Kits for Cell LinesCell Line Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 230Cell Line Optimization Kits for 4D Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 233Cell Line Kits for Nucleofector II /2b Device 234Cell Line Optimization Kit for Nucleofector II/2b Device237Nucleofector Kits for ParasitesBasic Parasite Nucleofector Kits 238Nucleofector Kit AccessoriesIntroduction240Nucleofector PLUS Supplements 241Mouse T Cell Nucleofector Medium 242pmaxCloning Vector 2436North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Table of Contents5 BioAssay Products and ServicesBioluminescent Cell HealthIntroduction248ViaLight Cell Proliferation and CytotoxicityBioAssay Kit 249ToxiLight Non-destructive Cytotoxicity BioAssay Kit 251Cell FunctionIntroduction254PDELight HTS cAMP Phosphodiesterase Assay Kit 255PPiLight Inorganic Pyrophosphate Assay 257AdipoRed Assay Reagent 259AdipoLyze Lipolysis Detection Assay 260OsteoAssay Human Bone Plate 261OsteoLyse Assay Kit 262OsteoImage Mineralization Assay 263BioAssay Accessory Products 2646 Electrophoresis and AnalysisNucleic Acid ElectrophoresisIntroduction268AgaroseAgarose Selection Guide 269SeaKem® LE Agarose 270MetaPhor Agarose 271The Highest Resolution Agarose Available 271NuSieve 3:1 Agarose 272NuSieve GTG Agarose 273SeaPlaque GTG Agarose 274SeaKem® GTG Agarose 275SeaPlaque Agarose 276ß-Agarase277SeaKem® Gold Agarose 278InCert Agarose and Megabase DNA Standards 279SeaKem® ME Agarose 280SeaPrep Agarose 280I.D.NA Agarose 281Precast Gels for DNA and RNA Selection Guide 282FlashGel System 283FlashGel System for DNA 283FlashGel System for Recovery 284FlashGel System for RNA 286FlashGel Camera 287FlashGel Specifications 287FlashGel SystemPower Supply 288Reliant Minigels 290Latitude HT Gels 292Latitude Midigels 294PAGEr Gold TBE Precast Gels 295Precast Gels and Related Products for RNA Analysis 296Markers, Stains and BuffersDNA Ladders and Markers 300GelStar® Nucleic Acid Gel Stain 302SYBR® Green Nucleic Acid Gel Stains 303AccuGENE Molecular Biology Buffers 304AccuGENE Electrophoresis Buffers 305Gel Support FilmsGelBond® Film 306GelBond® PAG Film 307GelBond® PAG Support Film 308Protein Electrophoresis and AnalysisIntroduction310Precast GelsPAGEr EX Protein Trial Kits 311PAGEr EX Gels 312ProSieveEX Stains 313Prosieve EX Running and Transfer Buffers 314PAGEr Gold Precast Gels 315Selecting the Best PAGEr Gold Precast Gel 316PAGEr Gold Scouting Kit 316PAGEr Minigel Chamber 317ProSieve Color Protein Markers 318ProSieve Protein Markers 319ProSieve ProTrack Dual Color Protein Loading Buffer 319AccuGENE Protein Electrophoresis Buffers 320ProSieve Blue Protein Staining Solution 320SYPRO® Protein Gel Stains 321SYPRO® Ruby Protein Gel Stain 322SYPRO® Red Protein Gel Stain 322SYPRO® Tangerine Protein Gel Stain 323SYPRO® Ruby Protein Blot Stain 323SYPRO® Protein Gel Stain Photographic Filter 324IsoGel Agarose and Precast IsoGel Agarose IEF Plates325IsoGel Agarose 325Precast IsoGel Agarose IEF Plates 326Agarose for Protein Separation 327Agarose for Protein Separation 328ProSieve 50 Acrylamide Gel Solution 329Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com7
7 ServicesCell ServicesIntroduction334Cells on Demand Cell Culture Services 336Cells on Demand Transfection Services 337Clonetics Conditionally Immortalized Cell Lines 338Pluripotent Stem Cell Services 339Testing ServicesQC Testing Solutions Services 342Endotoxin Detection Testing Services 343Mycoplasma Detection Testing Services 346Recertification Services 347Sterilization Services 348Genetically Modified Organism Testing Services 3498 QC Testing SolutionsEndotoxin DetectionIntroduction354Overview of LAL Testing Procedures 355Overview of Endotoxin Detection Methods 356Kinetic Chromogenic LAL Assay Overview 358Kinetic-QCL Kinetic Chromogenic LAL Assay 359Control Standard Endotoxin for Kinetic-QCLBulk Kinetic Chromogenic LAL 359QCL-1000 Endpoint Chromogenic LAL Assay 360Kinetic Turbidimetric LAL Assay Overview 361PYROGENT-5000 Kinetic Turbidimetric LAL Assay 362Reconstitution Buffer for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 362Control Standard Endotoxin for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 363Alternative Method – PyroGene RecombinantFactor C Assay 364PYROGENT Gel Clot LAL Assay Overview 367PYROGENT Gel Clot LAL Assay 368PYROGENT Plus Gel Clot LAL Assay 369PYROGENT Ultra Gel Clot LAL Assay 370PYROGENT Bulk Gel Clot LAL Assay 371Control Standard Endotoxin for PYROGENT Gel Clot LAL372Instrumentation and SoftwareELx808 Absorbance Plate Reader 374FLx800 Fluorescence Plate Reader 375PyroTec Liquid Handling System 376WinKQCL Endotoxin Detection and Analysis Software 377Accessory ProductsIntroduction380Test Tubes 380Sample Containers 381Plates381Pipette Tips 382Reservoirs382Dry Heat Block, Inserts and Vortex Mixer 383LAL Reagent Water 383β-G-Blocker384PYROSPERSE384MgCl 2 385Tris Buffer 385Endotoxin and Endotoxin Challenge Vials 3869 Technical InformationIntroduction390Primary Cell Culture and MediaOverview of Cell Culture Process forClonetics Human and Animal Cells* 391Safety Precautions <strong>with</strong> Clonetics Cells 392Media Preparation 392Clonetics and Poietics Cell Culture Media 394Pulmonary Epithelial Cell Media Options 395Endothelial Cell Media Options 396Fibroblast Cell Media Options 397Hepatocyte Media 397Keratinocyte Cell Media Options 398Mammary Epithelial Cell Media, Serum-free 399Melanocyte Cell Medium, Serum-free 399Neural Cell Medium, Low Serum 399Sertoli Cell Medium 399Prostate Epithelial Cell Medium, Serum-free 400Renal Cell Media, Low Serum 400Retinal Pigment Epithelial Cell Media 400Primary Neuron Growth Medium, Serum-free 400Human Mesenchymal Stem Cell Media 4018North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Rat Mesenchymal Stem Cell Media 401Human Adipose-Derived Stem Cells 402Preadipocyte Growth Medium 402Osteoclast Growth Medium 402Neural Progenitor Growth Medium 402Skeletal Cell Medium 403Skeletal Muscle Cell Medium 403Smooth Muscle Cell Medium 403Stromal Cell Medium, Low Serum 403Primary Cell MethodsCulture Set-up – Adherent Cell Types 405Thawing Cells – Adherent Cell Types 405Seeding – Adherent Cell Types 406Proliferating Cells – Adherent Cell Types 406Subculturing – Adherent Cell Types 407Subculturing into 96-well Plates 409Instructions for Cryopreservation 410Improving Cell Yield and Viability During Subculture 411Custom Cell Isolation Services 412Electrophoresis and AnalysisFrequently Asked Questions – Protein Analysis 458Agarose Types 460Preparation of Agarose Gels 461Loading Buffers 466Detection and Sizing of DNA in Agarose Gels 467Detecting DNA <strong>with</strong> GelStar®, SYBR® Green I or II NucleicAcid Gel Stains 470Detecting DNA <strong>with</strong> Ethidium Bromide 472Recovery of DNA from Agarose Gels 473Protein Separation in Polyacrylamide Gels 475Blotting Proteins from Polyacrylamide Gels 477Electrophoretic Theory 478Safety and Environmental Precautions 479Specific Chemical Hazards 479TransfectionCell Culture Tips for Cell Lines and Primary Cells Prior toTransfection413Important Vector Factors for Gene Expression 415Essentials for Preparing a Transfection Experiment <strong>with</strong>Plasmid DNA 419Guideline for Generation of Stable Cell Lines 420Designing an RNAi Experiment Using Nucleofection 427Media, Reagents and SeraCell Culture Technical Information 431Adaptation of Cell Cultures to Serum-free Medium 431Cryopreservation and Reconstitution 433Determination of Cell Numbers 434Powdered Media Preparation 436Subculturing Procedures for Mammalian Cells 437Formulations for BioWhittaker Classical Media 439Cell Culture Reagent Formulations 455Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com9
How to Use this <strong>Catalog</strong>This section explains some tools and hints we have includedin this catalog to help you find the information you need at aglance.IconsThe following icons identify or highlight certain types ofinformation. They guide you to additional product relatedinformation <strong>with</strong>in the catalog or on our website. And theytell you how to get Customer Service or Scientific Support.Technical Information SectionIn the back of this catalog, starting on page 387, you canfind the Technical Information and Sourcebook. This chapterholds additional technical information and FAQs on severalof our products included in this catalog. Please watch outfor this icon guiding you to the relevant page:Great additions to our expanding research productsportfolioAdditional product or technical information availableonlineContact our Customer Service or Scientific SupportRepresentatives for additional information or orderingassistanceOrdering information and instructionsProduct related scientific referencesAdditional technical information available in the chapter“Technical Information”. Please go to the catalog pagenumber indicatedStorage conditions10North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
How to OrderThere are several ways to order your products of choice <strong>with</strong>Lonza: Via our online shop, via e-mail, phone, fax or mail.Online OrderingOur website encompasses an extensive product portfolio.Along <strong>with</strong> the essential tools needed to manage yourelectronic purchases, you will find the process simple,secure and reliable. Our website offers:––Highest security standards––Ordering available 24 hours, 7 days a week––Latest technical, application, and safety information forour products––Custom prices––Sales tax, shipping charges, and expected shippingdates displayed before order is placed––Certificates of AnalysisTo order, you need a valid credit card (MasterCard®, VISA® orAmerican Express®) or purchase order, and a shippingaddress. Our system will always inform you of inventoryavailability as you add items to your shopping basket on thetop center of the website.Go to: http://www.lonza.com to access our online shop. Tofind your product of interest, please enter the catalognumber into the search field in the center of the webpage.Quick Order FormIt’s easy and saves time.If you already know the catalog numbers and quantities ofthe products you would like to purchase in our online shop,you may use the Quick Order Form (see screenshot)available at http://www.lonza.com/quickorderOrdering via Ariba or Other Third Party ProvidersWe support Ariba and other third party purchasing systemsthrough secure cXML communications. You have the choiceof purchasing via a pricelist (CIF electronic catalog) or usinga punch-out catalog system. We also accept and supportEDI standards 850 (purchase order) and 810 (salesinvoice), if your company conducts electronic transactionsusing EDI protocols.Please contact us if you would like to purchase productsusing any of these types of electronic transmissions.Direct Ordering via cXMLIf you have developed your own internal purchasing system,you can link directly to our order entry systems via securecXML communications protocols. Please contact us if youwould like to discuss using this type of system to place yourorders.For prompt response to your e-commerce questions, pleasee-mail us at ecommerce@lonza.com.Enter catalog number hereEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com11
How to OrderContinuedOrdering via E-mail, Phone, Fax or MailPlease include all of the following information on all of yourorders:––Purchase order number––Shipping address and billing address––Contact name and telephone number––Name and department of end user––Quote or reserve lot information––<strong>Catalog</strong> number, size, quantity, and name of productsorderedTo place an order <strong>with</strong> our Customer Service, please use anyof the following convenient options:North AmericaHours: Monday through Friday, 8:00am to 6:00pm ESTE-mail: order.us@lonza.comPhone: 800 638 8174Fax: 301 845 8338MailLonza Walkersville, Inc.Customer Service Department8830 Biggs Ford RoadWalkersville, MD 21793 USAEuropeHours:E-mail:Monday through Friday , 8:30am to 5:30pm CETorder.europe@lonza.comCustomer Service European Office NumbersPhoneFaxAustria 0800 201 538 0800 201 536Belgium +32 87 321 611 +32 87 321 634Denmark 808 83 159 808 83 178France 0800 91 19 81 0800 91 19 80Germany 0800 182 52 87 0800 182 52 78Ireland 1 800 654 253 1 800 654 259Italy 800 789 888 800 789 889Spain 900 963 298 900 963 299Sweden 020 790 220 020 797 710Switzerland 0800 83 86 20 0800 83 86 19The Netherlands 0800 022 4525 0800 022 4942United Kingdom 0808 2349 788 0808 2349 778MailLonza Verviers, S.p.r.l.Parc Industriel de Petit-Rechain4800 Verviers, BelgiumOrdering Information – Outside the U.S. and EuropePhoneFaxAustralia +61 3 9550 0883 +61 3 9550 0890E-mail: bioscience.australia@lonza.comBrazil +55 11 5641 3325 +55 11 2274 00 51E-mail: contact.br@lonza.comIndia +91 22 4342 4000 +91 22 4342 4050E-mail: scientificsupport.india@lonza.comJapan +81 3 5566 0612 +81 3 5566 0619E-mail: scientific.support.jp@lonza.comSingapore +65 6521 4379 +65 6521 4378E-mail: bioscience.asia@lonza.comIf your country is not listed above please check the currentlist of Lonza distributors on our website:www.lonza.com/distIn the event that you do not find an authorized distributorfor your country in the list, please contact:Lonza Walkersville, Inc.Customer Service Department8830 Biggs Ford RoadWalkersville, MD 21793 USAPhone: +1 301 898 7025Fax: +1 301 845 8338orLonza Verviers, S.p.r.l.Verviers, BelgiumPhone: +32 87 321 611Fax: +32 87 351 967E-mail: info.europe@lonza.comTerms and ConditionsPlease visit our website to find the current terms andconditions:www.lonza.com/termsandconditions12North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
How to Get Scientific SupportProviding world-class technical support for our products is atop priority. Valuable information is available to you aroundthe clock in our comprehensive online databases:––www.lonza.com/faqFrequently asked questions are answered online in ourknowledgebase––www.lonza.com/celldatabaseFind your cell of interest in our Cell Database––www.lonza.com/citationsThousands of scientific references can be viewed in ourCitation Database––www.lonza.com/technical-libraryThis Knowledge Center holds the latest technicalinformation, troubleshooting tips, and protocols onproducts and applications as well as full productinformation, instructions, Certificates of Analysis andMaterial Safety Data Sheets availableGet in Touch <strong>with</strong> Our Scientific Support Team by Phoneor E-mail:Scientific Support North AmericaHours: Monday through Friday, 8:00am to 6:00pm ESTPhone: 800 521 0390Fax: 301 845 8338E-mail: scientific.support@lonza.comPhone from outside the US: +1 301 898 7025Scientific Support InternationalHours: Monday through Friday; 9:00am and 6:00pm CETPhone: +49 221 99199 400E-mail: scientific.support.eu@lonza.comOur Scientific Support Representatives rely on years oflaboratory experience to assist <strong>with</strong> product selection andhelp you maximize product performance. Do you needadvice on which products suit your research projects? Doyou need support on a challenging experiment?Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com13
Trademarks and PatentsTrademarks of Lonza Group or its affiliates4D-Nucleofector96-well ShuttleABMAccuGENEAdipoLyzeAdipoRedAGMAlertAmniochromeB-ALIBEBMBEGMBioWhittakerBulletKitCalciFluorCBMCDMCells on DemandCGMCloneticsColorBurstEasyDiffEBMEGMFalconFBM-CDFBMFGM-CDFGMFlashGelFLx800GradeGTGHBMHCMHepsHL-1HMMInCertInsect-XPRESSIsoGelKaleidoscopeKBMKGM-CDKGMKinetic-QCLLatitudeLGM-3LymphochromemaxFPmaxGFPMBMMDEMEBMMEGMMetaPhorMGMMotorBlastMotorPlateMsBMMSCGM-CDMSCGMMsGMMycoAlertMycoZapNeuroBlastNHEPSNPBMNPMMNucleocuvetteNucleofectionNucleofectorNuSieveOBMOCPOGMOsteoAssayOsteoImageOsteoLysePAGErPBMPC-1PDELightPGMpmaxCloningpmaxFPpmaxGFPPNGMPoieticsPowerCHOPPiLightPrEBMPrEGMPro293ProCHOProDomaProDOMAProFreezeProNS0ProNSOProPerProSieveProTrackProVeroPyroGenePYROGENTPYROSPERSEPyroTecQCL-1000QuadColorRCGMReagentPackREBMREGMReliantRtEBMRtEGMS-ALISABMSAGMSCBMSCGMSeaPlaqueSeaPrepSeBMSeGMSelectShuttleSimplyLoadSingleQuotsSKBMSkBMSkGMSKGMSmallSmartStopSmBMSmGMSpeedFillStealthSunriseSynergySystemToxiLightTruBandUltraCHOUltraCULTUREUltraDOMA-PFUltraDOMAUltraGlutamineUltraMDCKUltraMEMUltraViaLightVialWinKQCLX-VIVOTrademarks from other companiesBD Falcon is a trademark of Becton, Dickinson and Company.BioTek, ELx808 and FLx800 are registered trademarks of BioTek Instruments,Inc.Eppendorf, Combtips and Biopur are registered trademarks of Eppendorf AG.GelStar, GelBond and SeaKem are trademarks of FMC Corp.Matrigel is a registered trademark of Discovery Labware, Inc.MYCOTOOL is a trademark of Roche.PER.C6 and PERMEXCIS are registered trademarks of Crucell Holland, BVRetronectin is a registered trademark of TaKaRa Shuzo Company, LTD.RNALater is a registered trademark of Ambion, Inc.Sephadex is a trademark of Amersham BioSciences.Sepharose is a registered trademark of Pharmacia Fine Chemicals, Inc.SpectraMax, Gemini and VersaMax are registered trademark of MolecularDevicesSYBR and SYPRO are registered trademarks of Molecular Probes Corporation,Inc.Transwell is a registered trademark of Data Packaging CorporationVersene is a registered trademark of Dow Chemical CompanyVortex-Genie is a registered trademark of Scientific Industries.All other trademarks mentioned herein are either the intellectual propertyof Lonza or belong to other companies where their related products wereused in the scope of research experiments or referenced scientificallyotherwise.PatentsLonza Group or its affiliates are owner or licensee** of the following patents.Products described herein may be covered by one or more of these UnitedStates patents, by pending patent applications, or by corresponding patentsor applications in other countries..U.S. Patents5,641,626** 6,512,236 6,365,341 6,117,2936,464,850 6,198,107 6,558,521 D510,7706,599,711 6,328,870 6,914,250 7,320,859D511,386 D524,449 5,486,359**7,332,332 5,385,839 6,905,585**European Patents:1297119 1383901 1390518 14765371525900 1522587 1702677 1741778PCT/EP01/07348 PCT/DE02/01489 PCT/DE02/0148314North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>11 Primary Cells and MediaClonetics Human Primary Cells and Media 27Clonetics Animal Primary Cells and Media 63Poietics Stem Cells and Media 75Poietics Immune Cells and Media 92
Primary Cells and Media1Primary Cells and MediaQuick Reference Guide 17Clonetics Human Primary Cells and MediaIntroduction28Bladder Cells and Media 29Cardiac Cells and Media 31Dermal Cells and Media 34Large Vessel Endothelial Cells and Media 37Microvascular Endothelial Cells and Media 39Gastrointestinal Cells and Media 42Lymphatic Cells and Media 43Mammary Epithelial Cells and Media 44Neural Cells and Media 45Ocular Cells and Media 46Pancreatic Islets 47Prostate Cells and Media 48Pulmonary Cells and Media 50Renal Cells and Media 54Reproductive Cells and Media 56Skeletal and Connective Tissue Cells and Media 58Skeletal Muscle Cells and Media 60Clonetics Animal Primary Cells and MediaIntroduction64Cardiac Cells and Media 65Fibroblasts Cells and Media 67Neural Cells and Media 68Ocular Cells and Media 71Skeletal Cells and Media 72Cell Culture Reagents 73Poietics Stem Cells and MediaIntroduction76Human Adipose-derived Stem Cells 77Human Bone Marrow Products 78Quadratox 4 Species Panel of Bone MarrowMononuclear Cells 78Mononuclear Cells 79Human CD34 + and CD133 + Progenitor Cells 80Human CD34 + Differentiation Supplements 81Human Neural Progenitor Cells and Media 82Human Osteoclast Precursor Cells and Media 83Human Pluripotent Stem Cells 84Human Preadipocyte Cells and Media 86Human Mesenchymal Stem Cells and Media 88Pluripotent Stem Cell Services 90Rat Mesenchymal Stem Cells and Media 91Poietics Immune Cells and MediaIntroduction93Human Dendritic Cells and Media 94Human CD14 + Monocytes and Media 95Human Natural Killer Cells96Human T Cells and Media 96These products are for research use only.Not approved for human or veterinary use, for application to humans or animals, or for use in clinical or in vivo procedures.WARNING: Clonetics AND Poietics products contain human source material, treat as potentially infectious.Each donor is tested and found non-reactive by an FDA approved method for the presence of HIV-1, Hepatitis B Virus, and Hepatitis C Virus.Where donor testing is not possible, cell products are tested for the presence of viral nucleic acid from HIV-1, Hepatitis B Virus, and Hepatitis C Virus. Testingcannot offer complete assurance that HIV-1, Hepatitis B Virus, and Hepatitis C Virus are absent. All human sourced products should be handled at the BiologicalSafety Level 2 to minimize exposure of potentially infectious products, as recommended in the CDC-NIH Manual, “Biosafety in Microbiological and BiomedicalLaboratories”, 5th ed. If you require further information, please contact your site Safety Officer or Scientific Support.Product Warranty – Cultures have a finite lifespan in vitro.Lonza guarantees the performance of its cells up to two years from purchase only if appropriate Clonetics or Poietics Media and Reagents are usedexclusively, and the recommended storage and use protocols are followed. Cell and media performance is not guaranteed if any modifications are made to thecomplete cell system.16North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Quick Reference GuideCell Type / Tissue Cell Cat. No. Recommended Medium Media Cat. No. PageAdventitial FibroblastsAorta CC-7014 SCGM CC-3205 321AstrocytesHuman Brain CC-2565 AGM CC-3186 45C57 Mouse Brain – Mixed M-AsM-330 AGM CC-3186 70CD1 Mouse Brain – Mixed M-AsM-430 AGM CC-3186 70Rat Brain Cx-Hi-Cp – Mixed R-AsM-530 AGM CC-3186 70Rat Brain – Cortex R-CxAs-520 AGM CC-3186 70Rat Brain – Hippocampus R-HiAs-521 AGM CC-3186 70Rat Brain – Striatum R-CpAs-522 AGM CC-3186 70BoneOsteoblasts CC-2538 OGM CC-3207 58Osteoclast Precursors 2T-110 OCP PT-8001 83Cardiac MyocytesRat Cardiac Myocytes R-CM-561 RCGM CC-4515 66ChondrocytesCartilage CC-2550 CGM CC-3216 58Dendritic CellsBlood CC-2701 LGM-3 CC-3211 93Endothelial CellsAorta CC-2535 EGM-2 CC-3162 32,38Aortic – Diabetes Type I CC-2919 EGM-2 CC-3162 32,38Aortic – Diabetes Type II CC-2920 EGM-2 CC-3162 32,38Coronary Artery CC-2585 EGM-2MV CC-3202 32,38Coronary Artery – Diabetes Type I CC-2921 EGM-2MV CC-3202 32,38Coronary Artery – Diabetes Type II CC-2922 EGM-2MV CC-3202 32,38Iliac Artery CC-2545 EGM-2MV CC-3202 38Pulmonary Artery CC-2530 EGM-2 CC-3162 38,52Umbilical Vein C2517A EGM-2 CC-3162 38Primary Cells and Media / Quick Reference GuideEndothelial Cells, PooledBovine Aorta BW-6002 EGM-MV CC-3125 66Umbilical Vein C2519A EGM-2 CC-3162 38Epithelial CellsBronchial / Tracheal (<strong>with</strong> Retinoic Acid) CC-2540S BEGM CC-3170 52Bronchial / Tracheal (<strong>with</strong>out Retinoic Acid) CC-2541 BEGM CC-3170 52Diseased Bronchial / Tracheal – COPD 00195275 BEGM CC-3170 51Diseased Bronchial / Tracheal – Asthma 00194911 BEGM CC-3170 51Kidney (Renal) CC-2556 REGM CC-3190 54Kidney (Renal) – Cortex CC-2554 REGM CC-3190 54Kidney (Renal) – Proximal Tubule CC-2553 REGM CC-3190 54More Quick Reference on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com17
Quick Reference GuideContinued1Cell Type / Tissue Cell Cat. No. Recommended Medium Media Cat. No. PageKidney (Renal) – Proximal Tubule – Diabetes Type II CC-2925 REGM CC-3190 54Mammary CC-2551 MEGM CC-3150 44Prostate CC-2555 PrEGM CC-3166 48,57Small Airway CC-2547 SAGM CC-3118 52Primary Cells and Media / Quick Reference GuideFibroblastsCardiac – Aortic CC-2903 FGM-3 CC-4526 32Cardiac – Ventricular CC-2904 FGM-3 CC-4526 32Dermal – Adult CC-2511 FGM-2 CC-3132 35Dermal – Neonatal CC-2509 FGM-2 CC-3132 35Diseased Lung (COPD) 00195277 FGM-2 CC-3132 51Diseased Lung (Asthma) 00194912 FGM-2 CC-3132Intestinal Myofibroblasts CC-2902 SmGM-2 CC-3182 42Lung CC-2512 FGM-2 CC-3132 52Periodontal Ligament CC-7049 SCGM CC-3205 58KeratinocytesEpidermal Adult– Diabetes Type II CC-2926 KGM-Gold 00192060 35Epidermal – Neonatal 00192907 KGM-Gold 00192060 35Epidermal – Neonatal, pooled 00192906 KGM-Gold 00192060 35MelanocytesNeonatal CC-2504 MGM-4 CC-3249 35Adult CC-2586 MGM-4 CC-3249 35Mesangial CellsKidney CC-2559 MsGM CC-3146 54Mesenchymal Stem CellsBone Marrow PT-2501 MSCGM PT-3001 87Microvascular Endothelial CellsBladder CC-7016 EGM-2MV CC-3202 29,40Blood CC-2815 EGM-2MV CC-3202 52Blood – Adult CC-2811 EGM-2MV CC-3202 35,40Blood – Neonatal CC-2813 EGM-2MV CC-3202 35,40Dermal – Adult CC-2543 EGM-2MV CC-3202 35,40Dermal Adult – Diabetes Type I CC-2929 EGM-2MV CC-3202 35,40Dermal Adult – Diabetes Type II CC-2930 EGM-2MV CC-3202 35,40Dermal – Neonatal CC-2505 EGM-2MV CC-3202 35,40Dermal – Neonatal, pooled CC-2516 EGM-2MV CC-3202 40Cardiac CC-7030 EGM-2MV CC-3202 32,40Cardiac – Diabetes Type I CC-2927 EGM-2MV CC-3202 32,40Cardiac – Diabetes Type II CC-2928 EGM-2MV CC-3202 32,40Lung CC-2527 EGM-2MV CC-3202 40,52Lymphatic CC-2814 EGM-2MV CC-3202 43,52Lymphatic – Adult CC-2810 EGM-2MV CC-3202 40,43Lymphatic – Neonatal CC-2812 EGM-2MV CC-3202 35,40,43Uterus CC-2564 EGM-2MV CC-3202 40,5718North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Quick Reference GuideContinuedMyoblastsSkeletal Muscle CC-2580 SkGM-2 CC-3245 61Skeletal Muscle Myoblasts – Diabetes Type I CC-2900 SkGM-2 CC-3245 61Skeletal Muscle Myoblasts – Diabetes Type II CC-2901 SkGM-2 CC-3245 611Neural Progenitor CellsBrain PT-2599 NPMM CC-3209 82NeuronsCD1 Mouse Brain – Cortex M-Cx-400 PNGM CC-4461 70CD1 Mouse Brain – Hippocampus M-Hi-401 PNGM CC-4461 70CD1 Mouse Brain – Striatum M-Cp-402 PNGM CC-4461 70C57 Mouse Brain – Cortex M-Cx-300 PNGM CC-4461 70C57 Mouse Brain – Striatum M-Cp-302 PNGM CC-4461 70Rat Brain – Cortex R-Cx-500 PNGM CC-4461 70Rat Brain – Striatum R-Cp-502 PNGM CC-4461 70Rat Brain – Hippocampus R-Hi-501 PNGM CC-4461 70Rat Brain – Hypothalamus R-HTh-507 PNGM CC-4461 70Rat Brain Cerebellum – Granule Cells R-CB-503 PNGM-A CC-4512 70Rat Embryo – Dorsal Root Ganglion R-eDRG-515 PNGM CC-4461 70Rat Spinal Cord – Dorsal Root Ganglion R-Drg-505 PNGM CC-4461 70Skeletal Muscle CellsSkeletal Muscle CC-2561 SkGM CC-3160 61Smooth Muscle CellsAorta CC-2571 SmGM-2 CC-3182 32Aorta– Diabetes Type I CC-2914 SmGM-2 CC-3182 32Aorta– Diabetes Type II CC-2916 SmGM-2 CC-3182 32Bladder CC-2533 SmGM-2 CC-3182 29Bronchial CC-2576 SmGM-2 CC-3182 52Coronary Artery CC-2583 SmGM-2 CC-3182 32Coronary Artery– Diabetes Type I CC-2917 SmGM-2 CC-3182 32Coronary Artery– Diabetes Type II CC-2918 SmGM-2 CC-3182 32Diseased Bronchial (COPD) 00195274 SmGM-2 CC-3182 51Diseased Bronchial (Asthma) 00194850 SmGM-2 CC-3182 51Prostate CC-2587 SmGM-2 CC-3182 48,57Pulmonary Artery CC-2581 SmGM-2 CC-3182 52Pulmonary Artery– Diabetes Type II CC-2913 SmGM-2 CC-3182 32Pulmonary Artery– Diabetes Type I CC-2915 SmGM-2 CC-3182 32Skeletal Muscle Myoblasts CC-2580 SkGM-2 CC-3245 61Umbilical Artery CC-2579 SmGM-2 CC-3182 57Uterus CC-2562 SmGM-2 CC-3182 57Primary Cells and Media / Quick Reference GuideStromal CellsProstate CC-2508 SCGM CC-3205 48,57More Quick Reference on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com19
Quick Reference GuideContinuedPart Number Proliferating Cell Type Description Recommended Media 6-well1Primary Cells and Media / Quick Reference GuideProliferating Cells – NormalCC-7014 AoAF – Human Aortic Adventitial Fibroblasts SCGM BulletKit CC-7014W6CC-2571 AoSMC – Human Aortic Smooth Muscle Cells SmGM-2 BulletKit CC-2571W6CC-2533 BdSMC – Human Bladder Smooth Muscle Cells SmGM-2 BulletKit CC-2533W6CC-2576 BSMC – Human Bronchial Smooth Muscle Cells SmGM-2 BulletKit CC-2576W6CC-2583 CASMC – Human Coronary Artery Smooth Muscle Cells SmGM-2 BulletKit CC-2583W6CC-2535 HAEC – Human Aortic Endothelial Cells EGM-2 BulletKit CC-2535W6CC-2535 HAEC – Human Aortic Endothelial Cells EGM BulletKit C2535AW6CC-2585 HCAEC – Human Coronary Artery Endothelial Cells EGM-2MV BulletKit CC-2585W6CC-2585 HCAEC – Human Coronary Artery Endothelial Cells EGM-MV BulletKit C2585AW6CC-2545 HIAEC – Human Iliac Artery Endothelial Cells EGM-2MV BulletKit CC-2545W6CC-2551 HMEC – Human Mammary Epithelial Cells MEGM BulletKit CC-2551W6CC-7016 HMVEC-Bd – Human Bladder Microvascular Endothelial Cells EGM-2MV BulletKit CC-7016W6CC-7030 HMVEC-C – Human Cardiac Microvascular Endothelial Cells EGM-2MV BulletKit CC-7030W6CC-2543 HMVEC-dAd – Human Dermal Microvascular Endothelial Cells – Adult EGM-2MV BulletKit CC-2543W6CC-2543 HMVEC-dAd – Human Dermal Microvascular Endothelial Cells – Adult EGM-MV BulletKit C2543AW6CC-2811 HMVEC-dBlAd – Human Dermal Blood Microvascular Endothelial Cells – Adult EGM-2MV BulletKit CC-2811W6CC-2813 HMVEC-dBlNeo – Human Dermal Blood Microvascular Endothelial Cells – Neonatal EGM-2MV BulletKit CC-2813W6CC-2810 HMVEC-dLyAd – Human Dermal Lymphatic Microvascular Endothelial Cells – Adult EGM-2MV BulletKit CC-2810W6CC-2812 HMVEC-dLyNeo – Human Dermal Lymphatic Microvascular Endothelial Cells –EGM-2MV BulletKit CC-2812W6Neonatal, Single DonorCC-2516 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells – Neonatal, Pooled EGM-2MV BulletKit CC-2516W6CC-2516 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells – Neonatal, Pooled EGM-MV BulletKit C2516AW6CC-2505 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells – Neonatal, Single Donor EGM-2MV BulletKit CC-2505W6CC-2505 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells – Neonatal, Single Donor EGM-MV BulletKit C2505AW6CC-2527 HMVEC-L – Human Lung Microvascular Endothelial Cells EGM-2MV BulletKit CC-2527W6CC-2814 HMVEC-Lly – Human Lung Lymphatic Microvascular Endothelial Cells EGM-2MV BulletKit CC-2814W6CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells EGM BulletKit C2530AW6CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells EGM-2 BulletKit CC-2530W6CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells EGM-2MV BulletKit C2530BW6CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells EGM-MV BulletKit C2530CW6CC-7049 HPdLF – Human Periodontal Ligament Fibroblasts SCGM BulletKit CC-7049W6CC-2554 HRCE – Human Renal Cortical Epithelial Cells REGM BulletKit CC-2554W6CC-2556 HRE – Human Renal Epithelial Cells REGM BulletKit CC-2556W6CC-2580 HSMM – Human Skeletal Muscle Myoblasts SkGM-2 BulletKit CC-2580W6CC-2519 HUVEC – Human Umbilical Vein Endothelial Cells, Pooled EGM BulletKit CC-2519W6C2519A HUVEC – Human Umbilical Vein Endothelial Cells, Pooled EGM-2 BulletKit C2519AW6C2517AS HUVEC – Human Umbilical Vein Endothelial Cells, Pooled, S-Part EGM-2 BulletKit C2519ASW6CC-2517 HUVEC – Human Umbilical Vein Endothelial Cells, Single Donor EGM BulletKit CC-2517W6C2517A HUVEC – Human Umbilical Vein Endothelial Cells, Single Donor EGM-2 BulletKit C2517AW6C2517AS HUVEC – Human Umbilical Vein Endothelial Cells, Single Donor, S-part EGM-2 BulletKit C2517ASW6CC-2550 NHAC-kn – Human Articular Chondrocytes CGM BulletKit CC-2550W6CC-2565 NHA – Human Astrocytes AGM BulletKit CC-2565W6CC-2540 NHBE – Human Bronchial /Tracheal Epithelial Cells BEGM BulletKit CC-2540W6CC-2541 NHBE – Human Bronchial /Tracheal Epithelial Cells BEGM BulletKit CC-2541W6CC-2511 NHDF-Ad – Human Dermal Fibroblasts – Adult FGM-2 BulletKit CC-2511W48CC-2509 NHDF-Neo – Human Dermal Fibroblasts – Neonatal FGM-2 BulletKit CC-2509W6CC-2501 NHEK-Ad – Human Epidermal Keratinocytes – Adult KGM BulletKit CC-2501W620North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Quick Reference GuideContinued12-well 24-well 48-well 96-well T-25 T-75 T-150 T-225CC-7014W12 CC-7014W24 CC-7014W48 CC-7014W96 CC7014T25 CC7014T75 CC7014T150 CC7014T225CC-2571W12 CC-2571W24 CC-2571W48 CC-0148 CC-2671 CC-0234 CC2571T150 CC2571T225CC-2533W12 CC-2533W24 CC-2533W48 CC-2533W96 CC-2533T25 CC-2533T75 CC2533T150 CC2533T225CC-2576W12 CC-2576W24 CC-2576W48 CC-0180 CC-2676 CC-0240 CC2576T150 CC2576T225CC-2583W12 CC-2583W24 CC-2583W48 CC-0096 CC-2683 CC-0258 CC2583T150 CC2583T225CC-2535W12 CC-2535W24 CC-2535W48 CC-0132 CC-2635 CC-0222 CC2535T150 CC2535T225C2535AW12 C2535AW24 C2535AW48 C2535AW96 C2535AT25 C2535AT75 C2535AT150 C2535AT225CC-2585W12 CC-2585W24 CC-2585W48 CC-0188 CC-2685 CC-0261 CC2585T150 CC2585T225C2585AW12 C2585AW24 C2585AW48 C2585AW96 C2585AT25 C2585AT75 C2585AT150 C2585AT225CC-2545W12 CC-2545W24 CC-2545W48 CC-0095 CC-2645 CC-0291 CC2545T150 CC2545T225CC-2551W12 CC-2551W24 CC-2551W48 CC-0140 CC-2651 CC-0228 CC2551T150 CC2551T225CC-7016W12 CC-7016W24 CC-7016W48 CC-7016W96 CC-7016T25 CC-7016T75 CC7016T150 CC7016T225CC-7030W12 CC-7030W24 CC-7030W48 CC-7030W96 CC-7030T25 CC-7030T75 CC-7030T150 CC-7030T225CC-2543W12 CC-2543W24 CC-2543W48 CC-2543W96 CC-2643 CC-0207 CC2543T150 CC2543T225C2543AW12 C2543AW24 C2543AW48 C2543AW96 C2543AT25 C2543AT75 C2543AT150 C2543AT225CC-2811W12 CC-2811W24 CC-2811W48 CC-2811W96 CC-2811T25 CC-2811T75 CC-2811T150 CC-2811T225CC-2813W12 CC-2813W24 CC-2813W48 CC-2813W96 CC-2813T25 CC-2813T75 CC-2813T150 CC-2813T225CC-2810W12 CC-2810W24 CC-2810W48 CC-2810W96 CC-2810T25 CC-2810T75 CC-2810T150 CC-2810T225CC-2812W12 CC-2812W24 CC-2812W48 CC-2812W96 CC-2812T25 CC-2812T75 CC-2812T150 CC-2812T225CC-2516W12 CC-2516W24 CC-2516W48 CC-2516W96 CC-2616 CC-0288 CC2516T150 CC2516T225C2516AW12 C2516AW24 C2516AW48 C2516AW96 C2516AT25 C2516AT75 C2516AT150 C2516AT225CC-2505W12 CC-2505W24 CC-2505W48 CC-0112 CC-2605 CC-0246 CC2505T150 CC2505T225C2505AW12 C2505AW24 C2505AW48 C2505AW96 C2505AT25 C2505AT75 C2505AT150 C2505AT225CC-2527W12 CC-2527W24 CC-2527W48 CC-0184 CC-2627 CC-0264 CC2527T150 CC2527T225CC-2814W12 CC-2814W24 CC-2814W48 CC-2814W96 CC-2814T25 CC-2814T75 CC-2814T150 CC-2814T225C2530AW12 C2530AW24 C2530AW48 C2530AW96 C2530AT25 C2530AT75 C2530AT150 C2530AT225CC-2530W12 CC-2530W24 CC-2530W48 CC-0128 CC-2630 CC-0219 CC2530T150 CC2530T225C2530BW12 C2530BW24 C2530BW48 C2530BW96 C2530BT25 C2530BT75 C2530BT150 C2530BT225C2530CW12 C2530CW24 C2530CW48 C2530CW96 C2530CT25 C2530CT75 C2530CT150 C2530CT225CC-7049W12 CC-7049W24 CC-7049W48 CC-7049W96 CC-7049T25 CC-7049T75 CC-7049T150 CC-7049T225CC-2554W12 CC-2554W24 CC-2554W48 CC-0172 CC-2654 CC-0270 CC2554T150 CC2554T225CC-2556W12 CC-2556W24 CC-2556W48 CC-2556W96 CC-2556T25 CC-2556T75 CC2556T150 CC2556T225CC-2580W12 CC-2580W24 CC-2580W48 CC-2580W96 CC-2580T25 CC-2580T75 CC2580T150 CC2580T225CC-2519W12 CC-2519W24 CC-2519W48 CC-2519W96 CC-2619 CC-0276 CC2519T150 CC2519T225C2519AW12 C2519AW24 C2519AW48 C2519AW96 C2519AT25 C2519AT75 C2519AT150 C2519AT225C2519ASW12 C2519ASW24 C2519ASW48 C2517ASW96 C2519AST25 C2519AST75 C2519AST150 C2519AST225CC-2517W12 CC-2517W24 CC-2517W48 CC-0124 CC-2617 CC-0216 CC2517T150 CC2517T225C2517AW12 C2517AW24 C2517AW48 C2517AW96 C2517AT25 C2517AT75 C2517AT150 C2517AT225C2517ASW12 C2517ASW24 C2517ASW48 C2517ASW96 C2517AST25 C2517AST75 C2517AST150 C2517AST225CC-2550W12 CC-2550W24 CC-2550W48 CC-2550W96 CC-2550T25 CC-2550T75 CC2550T150 CC2550T225CC-2565W12 CC-2565W24 CC-2565W48 CC-0093 CC-2665 CC-0297 CC2565T150 CC2565T225CC-2540W12 CC-2540W24 CC-2540W48 CC-0136 CC-2640 CC-0225 CC2540T150 CC2540T225CC-2541W12 CC-2541W24 CC-2541W48 CC-0100 CC-2641 CC-0285 CC2541T150 CC2541T225CC-0160 CC-2511W12 CC-2511W24 CC-2511W6 CC-2611 CC-0252 CC2511T150 CC2511T225CC-2509W12 CC-2509W24 CC-2509W48 CC-0116 CC-2609 CC-0210 CC2509T150 CC2509T225CC-2501W12 CC-2501W24 CC-2501W48 CC-0104 CC-2601 CC-0201 CC2501T150 CC2501T2251Primary Cells and Media / Quick Reference GuideEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com21
Quick Reference GuideContinued1Primary Cells and Media / Quick Reference GuidePart Number Proliferating Cell Type Description Recommended Media 6-wellProliferating Cells – NormalCC-2501A NHEK-Ad – Normal Human Epidermal Keratinocytes – Adult KGM-2 BulletKit C2501AW6192627 NHEK-Ad – Normal Human Epidermal Keratinocytes – Adult KGM-Gold BulletKit 00192627W6CC-2503 NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal KGM BulletKit CC-2503W6C2503A NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal KGM-2 BulletKit C2503AW6192907 NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal KGM-Gold BulletKit 192907W6CC-2507 NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal, Pooled KGM BulletKit CC-2507W6C2507A NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal, Pooled KGM-2 BulletKit C2507AW6192906 NHEK-Neo – Normal Human Epidermal Keratinocytes – Neonatal, Pooled KGM-Gold BulletKit 192906W6CC-2504 NHEM-Neo – Normal Human Epidermal Melanocytes – Neonatal MGM-4 BulletKit CC-2504W6CC-2512 NHLF – Normal Human Lung Fibroblasts FGM-2 BulletKit CC-2512W6CC-2559 NHMC – Normal Human Mesangial Cells MsGM BulletKit CC-2559W6CC-2538 NHOst – Normal Human Osteoblasts OGM BulletKit CC-2538W6CC-2581 PASMC – Human Pulmonary Artery Smooth Muscle Cells SmGM-2 BulletKit CC-2581W6CC-2555 PrEC – Human Prostate Epithelial Cells PrEGM BulletKit CC-2555W6CC-2508 PrSC – Human Prostate Stromal Cells SCGM BulletKit CC-2508W6CC-2587 PrSMC – Human Prostate Smooth Muscle Cells SmGM-2 BulletKit CC-2587W6CC-2553 RPTEC – Human Renal Proximal Tubule Cells REGM BulletKit CC-2553W6CC-2547 SAEC – Human Small Airway Epithelial Cells SAGM BulletKit CC-2547W6CC-2561 SkMC – Human Skeletal Muscle Cells SkGM BulletKit CC-2561W6CC-2579 UASMC – Human Umbilical Artery Smooth Muscle Cells SmGM-2 BulletKit CC-2579W6CC-2564 UtMVEC-Myo – Human Uterine Microvascular Endothelial Cells EGM-2MV BulletKit C2564AW6CC-2564 UtMVEC-Myo – Human Uterine Microvascular Endothelial Cells EGM-MV BulletKit CC-2564W6CC-2562 UtSMC – Human Uterine Smooth Muscle Cells SmGM-2 BulletKit CC-2562W6194987 H-RPE – Human Retinal Pigment Epithelial Cells RtEGM BulletKit 194987W6CC-2586 NHEM-Ad – Normal Human Melanocytes – Adult MGM-4 BulletKit CC-2586W6CC-2902 InMyoFib – Intestinal Myofibroblasts SmGM-2 BulletKit CC-2902W6CC-2903 NHCF-A – Normal Human Cardiac Fibroblasts – Atrial FGM-3 BulletKit CC-2903W6CC-2904 NHCF-V – Normal Human Cardiac Fibroblasts – Ventricular FGM-3 BulletKit CC-2904W622North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Quick Reference GuideContinued12-well 24-well 48-well 96-well T-25 T-75 T-150 T-225C2501AW12 C2501AW24 C2501AW48 C2501AW96 C2501AT25 C2501AT75 C2501AT150 C2501AT22500192627W12 00192627W24 00192627W48 00192627W96 00192627T25 00192627T75 00192627T150 00192627T225CC-2503W12 CC-2503W24 CC-2503W48 CC-0108 CC-2603 CC-0204 CC2503T150 CC2503T225C2503AW12 C2503AW24 C2503AW48 C2503AW96 C2503AT25 C2503AT75 C2503AT150 C2503AT225192907W12 192907W24 192907W48 192907W96 192907T25 192907T75 192907T150 192907T225CC-2507W12 CC-2507W24 CC-2507W48 CC-0156 CC-2607 CC-0255 CC2507T150 CC2507T225C2507AW12 C2507AW24 C2507AW48 C2507AW96 C2507AT25 C2507AT75 C2507AT150 C2507AT225192906W12 192906W24 192906W48 192906W96 192906T25 192906T75 192906T150 192906T225CC-2504W12 CC-2504W24 CC-2504W48 CC-2504W96 CC-2504T25 CC-2504T75 CC-2504T150 CC-2504T225CC-2512W12 CC-2512W24 CC-2512W48 CC2512T150 CC-2612 CC-0282 CC2512T225 CC-0164CC-2559W12 CC-2559W24 CC-2559W48 CC-0176 CC-2659 CC-0273 CC2559T150 CC2559T225CC-2538W12 CC-2538W24 CC-2538W48 CC-2538W96 CC-2538T25 CC-2538T75 CC2538T150 CC2538T225CC-2581W12 CC-2581W24 CC-2581W48 CC-0152 CC-2681 CC-0237 CC2581T150 CC2581T225CC-2555W12 CC-2555W24 CC-2555W48 CC-0088 CC-2655 CC-0310 CC2555T150 CC2555T225CC-2508W12 CC-2508W24 CC-2508W48 CC-2508W96 CC-2608 CC-2508T75 CC2508T150 CC2508T225CC-2587W12 CC-2587W24 CC-2587W48 CC-2587W96 CC-2587T25 CC-2587T75 CC2587T150 CC2587T225CC-2553W12 CC-2553W24 CC-2553W48 CC-0168 CC-2653 CC-0267 CC2553T150 CC2553T225CC-2547W12 CC-2547W24 CC-2547W48 CC-0094 CC-2647 CC-0294 CC2547T150 CC2547T225CC-2561W12 CC-2561W24 CC-2561W48 CC-0144 CC-2661 CC-0231 CC2561T150 CC2561T225CC-2579W12 CC-2579W24 CC-2579W48 CC-0192 CC-2679 CC-0243 CC2579T150 CC2579T225C2564AW12 C2564AW24 C2564AW48 C2564AW96 C2564AT25 C2564AT75 C2564AT150 C2564AT225CC-2564W12 CC-2564W24 CC-2564W48 CC-0084 CC-2644 CC-0331 CC2564T150 CC2564T225CC-2562W12 CC-2562W24 CC-2562W48 CC-0089 CC-2662 CC-0313 CC2562T150 CC2562T225194987W12 194987W24 194987W48 194987W96 194987T25 194987T75 194987T150 194987T225CC-2586W12 CC-2586W24 CC-2586W48 CC-2586W96 CC-2586T25 CC-2586T75 CC-2586T150 CC-2586T225CC-2902W12 CC-2902W24 CC-2902W48 CC-2902W96 CC-2902T25 CC-2902T75 CC-2902T150 CC-2902T225CC-2903W12 CC-2903W24 CC-2903W48 CC-2903W96 CC-2903T25 CC-2903T75 CC-2903T150 CC-2903T225CC-2904W12 CC-2904W24 CC-2904W48 CC-2904W96 CC-2904T25 CC-2904T75 CC-2904T150 CC-2904T2251Primary Cells and Media / Quick Reference GuideEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com23
Quick Reference GuideContinued1Primary Cells and Media / Quick Reference GuidePart Number Proliferating Cell Type Description Recommended Media 6-wellProliferating Cells – Diseased00194843 D-HLF-CF – Diseased Human Lung Fibroblasts – Cystic Fibrosis FGM-2 BulletKit 194843W600194850 D-BSMC-As – Diseased Bronchial Smooth Muscle Cells – Asthma SmGM-2 BulletKit 194850W600194911 D-HBE-As – Diseased Human Bronchial/Tracheal Epithelial Cells – Asthma BEGM BulletKit 194911W600194912 D-HLF-As – Diseased Human Lung Fibroblast Cells – Asthma FGM-2 BulletKit 194912W600195274 D-BSMC-COPD – Diseased Bronchial Smooth Muscle Cells – COPD SmGM-2 195274W600195275 D-HBE-COPD – Diseased Human Bronchial/Tracheal Epithelial Cells – COPD BEGM BulletKit 195275W600195277 D-HLF-COPD – Diseased Human Lung Fibroblast Cell – COPD FGM-2 BulletKit 195277W600196979 D-HBEC-CF – Diseased Human Bronchial/Tracheal Epithelial Cells – Cystic Fibrosis BEGM BulletKit 196979W600196980 D-HBSMC-CF – Diseased Human Bronchial Smooth Muscle Cells – Cystic Fibrosis SmGM-2 196980W6CC-2900 D-HSMM – Diseased Human Skeletal Muscle Myoblasts – Diabetes Type I SkGM-2 BulletKit CC-2900W6CC-2901 D-HSMM – Diseased Human Skeletal Muscle Myoblasts – Diabetes Type II SkGM-2 BulletKit CC-2901W6CC-2913 D-PASMC – Diseased Human Pulmonary Artery Smooth Muscle – Diabetes Type II SmGM-2 BulletKit CC-2913W6CC-2914 D-AoSMC – Diseased Human Aortic Smooth Muscle – Diabetes Type I SmGM-2 BulletKit CC-2914W6CC-2915 D-PASMC – Diseased Human Pulmonary Artery Smooth Muscle Cells – Diabetes Type I SmGM-2 BulletKit CC-2915W6CC-2916 D-AoSMC – Diseased Human Aortic Smooth Muscle – Diabetes Type II SmGM-2 BulletKit CC-2916W6CC-2917 D-CASMC – Diseased Human Coronary Artery Smooth Muscle – Diabetes Type I SmGM-2 BulletKit CC-2917W6CC-2918 D-CASMC – Diseased Human Coronary Artery Smooth Muscle – Diabetes Type II SmGM-2 BulletKit CC-2918W6CC-2919 D-HAEC – Diseased Human Aortic Endothelial – Diabetes Type I EGM-2 BulletKit CC-2919W6CC-2920 D-HAEC – Diseased Human Aortic Endothelial – Diabetes Type II EGM-2 BulletKit CC-2920W6CC-2921 D-HCAEC – Diseased Human Coronary Artery Endothelial Cells – Diabetes Type I EGM-2MV BulletKit CC-2921W6CC-2922 D-HCAEC – Diseased Human Coronary Artery Endothelial – Diabetes Type II EGM-2MV BulletKit CC-2922W6CC-2923 D-HPAEC – Diseased Human Pulmonary Artery Endothelial Cells – Diabetes Type I EGM-2 BulletKit CC-2923W6CC-2924 D-HPAEC – Diseased Human Pulmonary Artery Endothelial Cells – Diabetes Type II EGM-2 BulletKit CC-2924W6CC-2925 D-RPTEC – Diseased Human Renal Proximal Tubule Epithelial Cells – Diabetes Type II REGM BulletKit CC-2925W6CC-2926 D-HEK-Ad – Diseased Human Adult Epidermal Keratinocytes – Diabetes Type II KGM-Gold BulletKit CC-2926W6CC-2927 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells – Diabetes Type I EGM-2MV BulletKit CC-2927W6CC-2928 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells – Diabetes Type II EGM-2MV BulletKit CC-2928W6CC-2929 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cells – Diabetes Type I EGM-2MV BulletKit CC-2929W6CC-2930 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cells – Diabetes Type II EGM-2MV BulletKit CC-2930W6CC-2932 D-SAEC-As – Diseased Small Airway Epithelial Cells – Asthma BEGM BulletKit CC-2932W6CC-2933 D-SAEC – Diseased Small Airway Epithelial Cells – Cystic Fibrosis BEGM BulletKit CC-2933W6CC-2934 D-SAEC – Diseased Small Airway Epithelial Cells – COPD BEGM BulletKit CC-2934W624North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Quick Reference GuideContinued12-well 24-well 48-well 96-well T-25 T-75 T-150 T-225194843W12 194843W24 194843W48 194843W96 194843T25 194843T75 194843T150 194843T225194850W12 194850W24 194850W48 194850W96 194850T25 194850T75 194850T150 194850T225194911W12 194911W24 194911W48 194911W96 194911T25 194911T75 194911T150 194911T225194912W12 194912W24 194912W48 194912W96 194912T25 194912T75 194912T150 194912T225195274W12 195274W24 195274W48 195274W96 195274T25 195274T75 195274T150 195274T225195275W12 195275W24 195275W48 195275W96 195275T25 195275T75 195275T150 195275T225195277W12 195277W24 195277W48 195277W96 195277T25 195277T75 195277T150 195277T225196979W12 196979W24 196979W48 196979W96 196979T25 196979T75 196979T150 196979T225196980W12 196980W24 196980W48 196980W96 196980T25 196980T75 196980T150 196980T225CC-2900W12 CC-2900W24 CC-2900W48 CC-2900W96 CC-2900T25 CC-2900T75 CC-2900T150 CC-2900T225CC-2901W12 CC-2901W24 CC-2901W48 CC-2901W96 CC-2901T25 CC-2901T75 CC-2901T150 CC-2901T225CC-2913W12 CC-2913W24 CC-2913W48 CC-2913W96 CC-2913T25 CC-2913T75 CC-2913T150 CC-2913T225CC-2914W12 CC-2914W24 CC-2914W48 CC-2914W96 CC-2914T25 CC-2914T75 CC-2914T150 CC-2914T225CC-2915W12 CC-2915W24 CC-2915W48 CC-2915W96 CC-2915T25 CC-2915T75 CC-2915T150 CC-2915T225CC-2916W12 CC-2916W24 CC-2916W48 CC-2916W96 CC-2916T25 CC-2916T75 CC-2916T150 CC-2916T225CC-2917W12 CC-2917W24 CC-2917W48 CC-2917W96 CC-2917T25 CC-2917T75 CC-2917T150 CC-2917T225CC-2918W12 CC-2918W24 CC-2918W48 CC-2918W96 CC-2918T25 CC-2918T75 CC-2918T150 CC-2918T225CC-2919W12 CC-2919W24 CC-2919W48 CC-2919W96 CC-2919T25 CC-2919T75 CC-2919T150 CC-2919T225CC-2920W12 CC-2920W24 CC-2920W48 CC-2920W96 CC-2920T25 CC-2920T75 CC-2920T150 CC-2920T225CC-2921W12 CC-2921W24 CC-2921W48 CC-2921W96 CC-2921T25 CC-2921T75 CC-2921T150 CC-2921T225CC-2922W12 CC-2922W24 CC-2922W48 CC-2922W96 CC-2922T25 CC-2922T75 CC-2922T150 CC-2922T225CC-2923W12 CC-2923W24 CC-2923W48 CC-2923W96 CC-2923T25 CC-2923T75 CC-2923T150 CC-2923T225CC-2924W12 CC-2924W24 CC-2924W48 CC-2924W96 CC-2924T25 CC-2924T75 CC-2924T150 CC-2924T225CC-2925W12 CC-2925W24 CC-2925W48 CC-2925W96 CC-2925T25 CC-2925T75 CC-2925T150 CC-2925T225CC-2926W12 CC-2926W24 CC-2926W48 CC-2926W96 CC-2926T25 CC-2926T75 CC-2926T150 CC-2926T225CC-2927W12 CC-2927W24 CC-2927W48 CC-2927W96 CC-2927T25 CC-2927T75 CC-2927T150 CC-2927T225CC-2928W12 CC-2928W24 CC-2928W48 CC-2928W96 CC-2928T25 CC-2928T75 CC-2928T150 CC-2928T225CC-2929W12 CC-2929W24 CC-2929W48 CC-2929W96 CC-2929T25 CC-2929T75 CC-2929T150 CC-2929T225CC-2930W12 CC-2930W24 CC-2930W48 CC-2930W96 CC-2930T25 CC-2930T75 CC-2930T150 CC-2930T225CC-2932W12 CC-2932W24 CC-2932W48 CC-2932W96 CC-2932T25 CC-2932T75 CC-2932T150 CC-2932T225CC-2933W12 CC-2933W24 CC-2933W48 CC-2933W96 CC-2933T25 CC-2933T75 CC-2933T150 CC-2933T225CC-2934W12 CC-2934W24 CC-2934W48 CC-2934W96 CC-2934T25 CC-2934T75 CC-2934T150 CC-2934T2251Primary Cells and Media / Quick Reference GuideEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com25
Quick Reference GuideContinued1Primary Cells and Media / Quick Reference GuideCell Type – Blood Source – Bone Marrow Cord Blood Peripheral PageHematopoietic CellsFresh Bone Marrow 1M-125 78Mesenchymal Stem Cells PT-2501 87CD4 + T Cells 2W-200 95CD14 + Monocytes 2W-400A 94CD34 + Cells 2M-101 2C-101 80CD133 + Cells 2C-102 80Mononuclear Cells 2M-125A CC-2702 79, 93Stromal Cells 2M-302 78Products are available in various sizes. Please refer to the catalog for size information.Cat. No. Description Size PageHematopoietic Cell MediaPT-3926 HPGM Hematopoietic Progenitor Growth Medium 500 ml 81CC-3211 LGM-3 Lymphocyte Growth Medium-3 500 ml 93-9504-380Q X-VIVO 10 Chemically Defined, Serum-free Hematopoietic Cell Medium1 l 120<strong>with</strong> L-glutamine, gentamicin, and phenol red04-743Q X-VIVO 10 Chemically Defined, Serum-free Hematopoietic Cell Medium,1 l 120<strong>with</strong> L-glutamine, <strong>with</strong>out gentamicin or phenol red04-744Q X-VIVO 15 Chemically Defined, Serum-free Hematopoietic Cell Medium,1 l 120<strong>with</strong> L-glutamine, <strong>with</strong>out gentamicin or phenol redBE04-418FX-VIVO 15 Chemically Defined, Serum-free Hematopoietic Cell Medium,500 ml 120<strong>with</strong> gentamicin, L-glutamine, and phenol red04-418Q X-VIVO 15 Chemically Defined, Serum-free Hematopoietic Cell Medium,1 l 120<strong>with</strong> gentamicin, L-glutamine, and phenol red04-448Q X-VIVO 20 Chemically Defined, Serum-free Hematopoietic Cell Medium,<strong>with</strong> gentamicin, L-glutamin e, and phenol red1 l 12026North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Clonetics Human Primary Cells and MediaIn vivo relevance. In vitro results.1Clonetics Human Primary Cells and MediaIntroduction28Bladder Cells and Media 29Cardiac Cells and Media 31Dermal Cells and Media 34Large Vessel Endothelial Cells and Media 37Microvascular Endothelial Cells and Media 39Gastrointestinal Cells and Media 42Lymphatic Cells and Media 43Mammary Epithelial Cells and Media 44Neural Cells and Media 45Ocular Cells and Media 46Pancreatic Islets 47Prostate Cells and Media 48Pulmonary Cells and Media 50Renal Cells and Media 54Reproductive Cells and Media 56Skeletal and Connective Tissue Cells and Media 58Skeletal Muscle Cells and Media 60Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com27
Introduction1Primary Cells and Media / Clonetics Human Primary Cells and MediaClonetics Human Primary Cells and Media include cellsthat are primarily derived from normal human tissue andtest negative for HIV-1, Hepatitis B and C, mycoplasma andsterility. Immuno and special staining protocols, as well ascharacteristic morphology are used to characterize thecells and authenticate their identity. A Certificate of Analysisis available for each cryopreserved cell type and lot. Thecells perform when used <strong>with</strong> the optimized systemcomprised of Clonetics Cells, Media, Reagents, andProtocol. Most cells are available cryopreserved,proliferating (in either flasks or plates), or as pellets inRNALater®, a reagent that inactivates RNases and stabilizesRNA <strong>with</strong>in unfrozen tissues and cells.Clonetics Media Kits have been specially designed tosupport the growth of these cells. These Media BulletKitsare comprised of basal media and SingleQuots Kits ofgrowth factors and supplements. For detailed informationabout these media systems, please See pages 395–404.■■General Cell and Media Information––Proliferating cells are offered in the following formats,Flasks (T-25, T-75, T-150, T-225) and Multiwell Plates(6, 12, 24, 48, and 96-wells). Contact Customer Serviceor Scientific Support for order placement, deliveryschedules, and any other questions regardingalternative formats for cell culture reagents––Cell pellets in RNALater® are available as well <strong>with</strong>10 million cells/pellet, contact Customer Service fororder placement––Clonetics Cells are guaranteed to perform to ourrelease criteria when cultured <strong>with</strong> the provided protocolin our recommended media and reagents––Media systems are offered as BulletKits (basalmedium and SingleQuots Kit) to provide the flexibilityto manipulate media components specific to yourapplication, and a longer shelf life prior to useGeneral Ordering and Shipping InformationCryopreserved cells and media products are normallyshipped Monday – Thursday for next day delivery.Saturday and Monday deliveries are available uponspecial request.Proliferating cell orders must be placed no later thanTuesday at 5:00 pm EST for shipment the followingMonday (for delivery on Tuesday). Orders placed afterTuesday will be held until the next production cycle.When you place your order for proliferating cells, pleaseorder the appropriate media. Other cell types may beavailable upon request. Well plate orders require anadditional passage and take an extra week.Cell pellet orders require 7–10 production days. Pleaseplan accordingly.28North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bladder Cells and MediaThe bladder serves as a reservoir for water solublebyproducts generated during cell metabolism. Solublewastes are excreted through the urinary system, whichconsists of the kidneys, ureters, urinary bladder, andurethra.1■■Source––Human bladder smooth muscle cells and humanbladder microvascular endothelial cells both isolatedfrom specific tissues layers surrounding the bladder––■■Applications––Overactive bladder––Cancer––Urologic disease■■Cell Testing and Specifications––BdSMC stain positive for smooth muscle α-actin andnegative for von Willebrand Factor––HMVEC-Bd stain positive for von Willebrand Factor andLDL and negative for smooth muscle α-actin––Both cell types are guaranteed through ten populationdoublings when using Clonetics Media and ReagentsCell Type Description RecommendedMediaHMVEC-Bd culture stained for von Willebrand FactorHMVEC-Bd at >90% confluencyCryopreservedCellsProliferating CellsRecommendedSeeding DensityTime toSubcultureBdSMC Bladder smooth muscle SmGM-2 BulletKit 3rd passage 4th passage 3,500 cells/cm 2 6 to 9 daysHMVEC-Bd Bladder microvascularEGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 6 to 9 daysendothelialOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2533 CC-2533 BdSMC – Human Bladder Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-7016 CC-7016 HMVEC-Bd – Human Bladder Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Primary Cells and Media / Clonetics Human Primary Cells and MediaMore ordering information on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com29
Bladder Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3182 CC-3182 SmGM-2 Smooth Muscle Growth Medium -2 BulletKit Includes basal medium and SingleQuots Kit KitCC-4149 CC-4149 SmGM-2 Smooth Muscle Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3182/6 SmGM-2 Smooth Muscle Cell Growth Medium-2 BulletKit Six pack, includes basal medium and SingleQuots Kit KitCC-3181 CC-3181 SmBM Smooth Muscle Cell Basal Medium 500 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, and 100 ml eachHEPES buffered saline solutionCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth FactorsCC-3202/6 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 Six pack, includes basal medium and SingleQuots Kit KitBulletKitCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 Includes basal medium and SingleQuots KitKitBulletKitCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlSee pages 395–404.Related ProductsCell Culture Reagents 138Nucleofector Kits for Primary Smooth Muscle Cells 214Nucleofector Kits for Primary Endothelial Cells 202Page30North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cardiac Cells and MediaCardiac cells are used to study the functions and generalpathophysiology of the human cardiovascular system.Some of these cell types are available from normal, Type Iand Type II diabetic donors.1■■Source––Human aorta adventitial fibroblasts isolated from tunicaexternal of ascending or descending aorta––Cardiac fibroblasts isolated from atrial and ventricularcardiac tissue––Endothelial cells isolated from human aorta, coronaryartery and small vessel endothelial cells from ventricletissue––Smooth muscle cells isolated from aorta and coronaryartery■■Applications––Arrhythmia––Cardiomyopathy––Heart failure––Preventative cardiology––Vascular research■■Cell Testing and Specifications––Endothelial cells – Positive for acetylated low densitylipoprotein LDL uptake, normal healthy endothelial cellstake LDL and store in vacuoles; positive for vonWillebrand Factor Expression/Factor VIII, positive stainreflects a healthy endothelial cell and von WillebrandFactor synthesis is critical to blood clotting; up to 15population doublings guaranteed when using CloneticsMedia and Reagents––Fibroblasts – Cardiac fibroblasts stain positive forcollagen I and negative for von Willebrand factor VIII andare guaranteed through five population doublings whenusing Clonetics Media and Reagents, AoAF stainnegative for α-actin and are guaranteed through tenpopulation doublings when using Clonetics Media andReagentsHCAEC culture stained for von Willebrand FactorHCAEC culture stained for von Willebrand FactorHuman cardiac fibroblasts (ventricle) at fifth passage stained for collagenand counterstained <strong>with</strong> DAPI (20x)––Smooth muscle cells – Stain positive for α-actin andnegative for von Willebrand Factor after differentiation,and are guaranteed through 15 population doublingswhen using Clonetics Media and ReagentsPrimary Cells and Media / Clonetics Human Primary Cells and MediaCell Type Description Recommended Media CryopreservedCellsProliferating CellsRecommended SeedingDensityTime toSubcultureHAEC* Aortic endothelial EGM-2 BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHCAEC * Coronary artery EGM-2MV BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHMVEC-C * Cardiac microvascular EGM-2MV BulletKit 3rd passage n/a 2,500–5,000 cells/cm 2 5 to 9 daysAoAF Aortic adventitial fibroblasts SCGM BulletKit 2nd passage 3rd passage 3,500 cells/cm 2 6 to 9 daysNHCF-A Atrial cardiac fibroblasts FGM-3 BulletKit 2nd passage 3rd passage 5,000 cells/cm 2 6 to 9 daysNHCF-V* Ventricle cardiac fibroblasts FGM-3 BulletKit 2nd passage 3rd passage 5,000 cells/cm 2 6 to 9 daysAoSMC * Aortic smooth muscle SmGM-2 BulletKit 3rd passage 4th or 5th passage 3,500 cells/cm 2 6 to 10 daysCASMC * Coronary artery SmGM-2 BulletKit 3rd passage 4th or 5th passage 3,500 cells/cm 2 6 to 10 daysHPAEC* Pulmonary Artery Endothelial CC-2530 EGM-2 3rd passage 4th passage 2500–5000 cells/cm 2 5 to 9 daysPASMC* Pulmonary Artery Smooth Muscle SmGM-2 3rd passage 4th or 5th passage 3500 cells/cm 2 6 to 10 days* Cells also available from Type I and Type II diabetic donorsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com31
Cardiac Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeNormal CellsCC-2535 CC-2535 HAEC – Human Aortic Endothelial Cells Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialCC-2585 CC-2585 HCAEC – Human Coronary Artery Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-7030 CC-7030 HMVEC-C – Human Cardiac Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-7014 CC-7014 AoAF – Human Aortic Adventitial Fibroblasts Cryopreserved, in SCGM BulletKit ≥500,000 cells/vialCC-2903 CC-2903 NHCF-A – Normal Human Artial Cardiac Fibroblasts Cryopreserved, in FGM-3 BulletKit ≥500,000 cells/vialCC-2904 CC-2904 NHCF-V – Normal Human Ventricular Cardiac Fibroblasts Cryopreserved, in FGM-3 BulletKit ≥500,000 cells/vialCC-2571 CC-2571 AoSMC – Human Aortic Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2583 CC-2583 CASMC – Human Coronary Artery Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialDiseased CellsCC-2919 CC-2919 D-HAEC – Diseased Human Aortic Endothelial – Diabetes Type I Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialCC-2920 CC-2920 D-HAEC – Diseased Human Aortic Endothelial – Diabetes Type II Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialCC-2921 CC-2921 D-HCAEC – Diseased Human Coronary Artery Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type ICC-2922 CC-2922 D-HCAEC – Diseased Human Coronary Artery Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type IICC-2923 CC-2923 D-HPAEC – Diseased Human Pulmonary Artery Endothelial Cells – Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialDiabetes Type ICC-2924 CC-2924 D-HPAEC – Diseased Human Pulmonary Artery Endothelial Cells – Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialDiabetes Type IICC-2927 CC-2927 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type ICC-2928 CC-2928 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type IICC-2914 CC-2914 D-AoSMC – Diseased Human Aortic Smooth Muscle – Diabetes Type I Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2916 CC-2916 D-AoSMC – Diseased Human Aortic Smooth Muscle – Diabetes Type II Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2917 CC-2917 D-CASMC – Diseased Human Coronary Artery Smooth Muscle – Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialDiabetes Type ICC-2918 CC-2918 D-CASMC – Diseased Human Coronary Artery Smooth Muscle – Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialDiabetes Type IICC-2915 CC-2915 D-PASMC – Diseased Human Pulmonary Artery Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vial– Diabetes Type ICC-2913 CC-2913 D-PASMC – Diseased Human Pulmonary Artery Smooth Muscle Cells– Diabetes Type IICryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vial32North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cardiac Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-3162 CC-3162 EGM-2 Endothelial Cell Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-4176 CC-4176 EGM-2 Endothelial Cell Growth Medium-2 SingleQuots SupplementsKitand Growth FactorsCC-3131 CC-3131 FBM Fibroblast Basal Medium 500 mlCC-4525 CC-4525 FGM-3 Cardiac Fibroblast Growth Medium-3 SingleQuotsKitSupplements and Growth FactorsCC-4526 CC-4526 FGM-3 Cardiac Fibroblast Growth Medium-3 BulletKit Includes growth basal medium, differentiation Kitbasal media and SingleQuots Kit, only sold asBulletKit MediumCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth FactorsCC-3181 CC-3181 SmBM Smooth Muscle Cell Basal Medium 500 mlCC-3182 CC-3182 SmGM-2 Smooth Muscle Growth Medium -2 BulletKit Includes basal medium and SingleQuots Kit KitCC-4149 CC-4149 SmGM-2 Smooth Muscle Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3204 CC-3204 SCBM Stromal Cell Basal Medium 500 mlCC-3205 CC-3205 SCGM Stromal Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4181 CC-4181 SCGM Stromal Cell Growth Medium SingleQuots Supplements andKitGrowth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, and 100 ml eachHEPES buffered saline solutionNOTE: Normal cell media is recommended for related disease cell types.Related ProductsRat AoSMC and Cardiomyocytes 32Nucleofector Kits for Human Coronary Artery Endothelial Cells (HCAEC) 202Nucleofector Kits for Mammalian Endothelial Cells 205Nucleofector Kits for Mammalian Fibroblasts 211Nucleofector Kits for Human Aortic Smooth Muscle Cells 214Page1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com33
Dermal Cells and MediaContinuedCell Type Description RecommendedMediaCryopreservedCellsProliferating CellsRecommendedSeeding DensityTime toSubcultureHMVEC-dAd Adult dermal microvascular EGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysHMVEC-dBIAd Adult dermal bloodEGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 4 to 7 daysmicrovascularHMVEC-dBINeo Neonatal dermal bloodEGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 4 to 7 daysmicrovascularHMVEC-dLyAd Adult dermal lymphaticEGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 4 to 7 daysmicrovascularHMVEC-dNeo Neonatal dermal microvascular EGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysHMVEC-dLyNeo Neonatal dermal lymphatic EGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 4 to 7 daysmicrovascularNHDF-Ad Adult dermal fibroblasts FGM-2 BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 daysNHDF-Neo Neonatal dermal fibroblasts FGM-2 BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 daysNHEK-Ad Epidermal keratinocytes, adult KGM-Gold BulletKit 1st passage 2nd passage 3,500 cells/cm 2 5 to 9 daysNHEK-Neo Epidermal keratinocytes,KGM-Gold BulletKit 1st passage 2nd passage 3,500 cells/cm 2 5 to 9 daysneonatalNHEK-Neo Epidermal keratinocytes,KGM-Gold BulletKit 1st passage 2nd passage 3,500 cells/cm 3 6 to 9 daysPooledneonatal, pooledNHEM-Neo Neonatal normal humanepidermal melanocytesMGM-4 BulletKit 3rd passage 4th passage 10,00 cells/cm 2 9 to 14 daysNHEM-AdAdult normal humanepidermal melanocytesMGM-4 BulletKit+ ET-3 Supplement2nd passage 3rd passage 10,000 cells/cm 2 9 to 14 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeDiseased CellsCC-2926 CC-2926 D-HEK-Ad – Diseased Human Adult Epidermal Keratinocytes – Cryopreserved, in KGM-Gold BulletKit ≥500,000 cells/vialDiabetes Type IICC-2929 CC-2929 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCells – Diabetes Type ICC-2930 CC-2930 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCells – Diabetes Type IINormal CellsCC-2505 CC-2505 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells, Cryopreserved, in EGM-2MV BulletKit, ≥500,000 cells/vialneonatalsingle donorCC-2543 CC-2543 HMVEC-dAd – Human Dermal Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vial– AdultCC-2811 CC-2811 HMVEC-dBlAd – Human Dermal Blood Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCells – AdultCC-2812 CC-2812 HMVEC-dLyNeo – Human Dermal Lymphatic Microvascular Cryopreserved, in EGM-2MV BulletKit, ≥500,000 cells/vialEndothelial Cells – Neonatalsingle donorCC-2813 CC-2813 HMVEC-dBl-Neo – Human Dermal Blood MicrovascularCryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialEndothelial Cells – NeonatalCC-2509 CC-2509 NHDF-Neo – Normal Human Dermal Fibroblasts – Neonatal Cryopreserved, in FGM-2 BulletKit ≥500,000 cells/vialCC-2511 CC-2511 NHDF-Ad – Normal Human Dermal Fibroblasts – Adult Cryopreserved, in FGM-2 BulletKit ≥500,000 cells/vial192627 192627 NHEK-Ad – Normal Human Epidermal Kerationocytoes – Adult Cryopreserved, in KGM-Gold BulletKit ≥500,000 cells/vial1Primary Cells and Media / Clonetics Human Primary Cells and Media00192906 00192906 NHEK-Neo – Normal Human Epidermal Keratinocytes –Neonatal00192907 00192907 NHEK-Neo – Normal Human Epidermal Keratinocytes –NeonatalCryopreserved, in KGM-Gold BulletKit,pooledCryopreserved, in KGM-Gold BulletKit≥500,000 cells/vial≥500,000 cells/vialCC-2504 CC-2504 NHEM-Neo – Normal Human Epidermal Melanocytes – Neonatal Cryopreserved, in MGM-4 BulletKit ≥500,000 cells/vialCC-2586 CC-2586 NHEM-Ad – Normal Human Epidermal Melanocytes – Adult Cryopreserved, in MGM-4 BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com35
Dermal Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description Size10-547F BE10-547F Hank‘s Buffered Saline Solution Without phenol red, calcium or magnesium 500 mlCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 Includes basal medium and SingleQuots KitKitBulletKitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth FactorsCC-3131 CC-3131 FBM Fibroblast Basal Medium 500 mlCC-3132 CC-3132 FGM-2 Fibroblast Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-4126 CC-4126 FGM-2 Fibroblast Growth Medium-2 SingleQuotsKitSupplements and Growth Factors00192060 00192060 KGM-Gold Keratinocyte Growth Medium BulletKit Includes growth basal medium, differentiation basal media Kitand SingleQuots Kit, only sold as BulletKit Medium00192151 00192151 KBM-Gold Keratinocyte Basal Medium 500 ml00192152 00192152 KGM-Gold Keratinocyte Growth Medium SingleQuotsKitSupplements and Growth Factors00195769 00195769 KGM-Gold Keratinocyte Growth Medium BulletKit Calcium-free, includes basal medium and SingleQuots Kit Kit00195130 00195130 KBM-Gold Keratinocyte Basal Medium Calcium-free, phenol red-free 500 mlCC-4455 CC-4455 TheraPEAK KGM-CD Keratinocyte Growth MediumChemically defined, includes basal medium and SingleQuots KitBulletKitKitCC-4456 CC-4456 KGM-CD Keratinocyte Growth Medium SingleQuotsChemically definedKitSupplements and Growth FactorsCC-3255 CC-3255 KBM-CD Keratinocyte Basal Medium Chemically defined 500 mlCC-3249 CC-3249 MGM-4 Melanocyte Growth Medium-4 BulletKit Includes basal medium and SingleQuots Kit KitCC-3250 CC-3250 MBM-4 Melanocyte Basal Medium-4 500 mlCC-4435 CC-4435 MGM-4 Melanocyte Growth Medium-4 SingleQuotsKitSupplements and Growth Factors17-516F BE17-516F Phosphate Buffered Saline (1X) 6.7 mM (PO4) <strong>with</strong>out calcium or magnesium 500 mlCC-5012 CC-5012 Trypsin/EDTA Solution 100 ml17-711E BE17-711E Versene® (EDTA), 0.02% 0.2 g/l Ethylenediaminetetraacetic acid (0.53 mM) in DPBS, 100 ml<strong>with</strong>out calcium or magnesiumCC-5002 CC-5002 Trypsin Neutralizing Solution 100 mlCC-4510 CC-4510 Endothelin-3 (ET-3) Growth Supplement 130 μgSee pages 395–404.Related ProductsEndothelial cells must be cultured in their isolation medium for best results.Nucleofector Kits for Primary Dermal Cells 200Nucleofector Kits for Primary Endothelial Cells 202Nucleofector Kits for Primary Fibroblasts 209Page36North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Large Vessel Endothelial Cells and MediaEndothelial cells line the inside surface of blood vessels,heart, lymphatic vessels, body cavities and other organs ofnormal human tissue.We offer many of these cell types from normal, Type I andType II diabetic donors.1■■Source––Large vessel endothelial cells are isolated from thehuman aorta, umbilical artery and vein, and coronary,iliac and pulmonary arteries■■Applications––Atherosclerosis––Arteriosclerosis––Drug uptake or drugdiscovery––Wound healing––Angiogenesis––Inflammation––Oncology■■Cell Testing and Specifications––Endothelial cells – Test positive for acetylated lowdensity lipop rotein uptake; positive for von WillebrandFactor Expression/Factor VIII. HUVEC test ≥90% doublepositive for CD31/CD105 markers by flow cytometry. Upto 15 population doublings are guaranteed <strong>with</strong> normalcells when using Clonetics Media and ReagentsPrescreened HUVECs are isolated in EGM-2 medium,pooled from 3 to 5 donors, and tested for angiogenesis/endothelial health related markers: Axl, eNOS, Tie-2, andVEGFr2.Cell Type Description RecommendedMediaCryopreservedCellsHCAEC culture stained for von Willebrand FactorHAEC at >90% confluencyHUVEC stained for von Willebrand Factor and counterstained <strong>with</strong> DAPIProliferating CellsRecommended SeedingDensityTime toSubcultureHAEC Aortic endothelial EGM-2 BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHCAEC Coronary artery EGM-2MV BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHIAEC Iliac artery endothelial EGM-2MV BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHPAEC Pulmonary artery EGM-2 BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHUAEC Umbilical artery EGM-MV BulletKit 3rd passage 4th passage 2,500–5,000 cells/cm 2 5 to 9 daysHUVEC Umbilical vein EGM-2 or EGM BulletKit 1st passage 2nd passage 2,500–5,000 cells/cm 2 5 to 9 daysHUVEC-XL Umbilical vein EGM-2 BulletKit 3rd passage n/a 2,500–5,000 cells/cm 2 5 to 9 daysPrimary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com37
Large Vessel Endothelial Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeDiseased CellsCC-2919 CC-2919 D-HAEC – Diseased Human Aortic Endothelial – Cryopreserved, in EGM-2 BulletKit≥500,000 cells/vialDiabetes Type ICC-2920 CC-2920 D-HAEC – Diseased Human Aortic Endothelial – Cryopreserved, in EGM-2 BulletKit≥500,000 cells/vialDiabetes Type IICC-2921 CC-2921 D-HCAEC – Diseased Human Coronary ArteryCryopreserved, in EGM-2MV BulletKit≥500,000 cells/vialEndothelial Cells – Diabetes Type ICC-2922 CC-2922 D-HCAEC – Diseased Human Coronary ArteryCryopreserved, in EGM-2MV BulletKit≥500,000 cells/vialEndothelial Cells – Diabetes Type IICC-2923 CC-2923 D-HPAEC – Diseased Human Pulmonary ArteryCryopreserved, in EGM-2 BulletKit≥500,000 cells/vialEndothelial Cells – Diabetes Type ICC-2924 CC-2924 D-HPAEC – Diseased Human Pulmonary ArteryCryopreserved, in EGM-2 BulletKit≥500,000 cells/vialEndothelial Cells – Diabetes Type IINormal CellsCC-2535 CC-2535 HAEC – Human Aortic Endothelial Cells Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialCC-2585 CC-2585 HCAEC – Human Coronary Artery Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-2545 CC-2545 HIAEC – Human Iliac Artery Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-2530 CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialC2517A C2517A HUVEC – Human Umbilical Vein Endothelial Cells Cryopreserved, in EGM-2 BulletKit, single donor ≥500,000 cells/vialC2517AS C2517AS HUVEC – Human Umbilical Vein Endothelial Cells Pre-screened, in EGM-2, single donor ≥500,000 cells/vialC2519A C2519A HUVEC – Human Umbilical Vein Endothelial Cells Cryopreserved, in EGM-2 BulletKit, pooled ≥500,000 cells/vialC2519AS C2519AS HUVEC – Human Umbilical Vein Endothelial Cells Pre-screened, in EGM-2, pooled donor ≥500,000 cells/vialCC-2517 CC-2517 HUVEC – Human Umbilical Vein Endothelial Cells Cryopreserved, in EGM-2 BulletKit, single donor ≥500,000 cells/vialCC-2519 CC-2519 HUVEC – Human Umbilical Vein Endothelial Cells Cryopreserved, in EGM-2 BulletKit, pooled ≥500,000 cells/vial00191027 00191027 HUVEC-XL – Human Umbilical Vein Endothelial Cells Cryopreserved, in EGM-2 BulletKit, expanded, pooled ≥10 million cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3162 CC-3162 EGM-2 Endothelial Cell Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-4176 CC-4176 EGM-2 Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth FactorsCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Includes basal medium and SingleQuots KitKitMedium-2 BulletKitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell GrowthKitMedium-2 SingleQuots Supplements and GrowthFactorsCC-3024 CC-3024 EGMComplete Endothelial Cell Growth Medium With 2% FBS 500 mlCC-3121 CC-3121 EBM Endothelial Cell Basal Medium 500 mlCC-4133 CC-4133 EGM Endothelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3125 CC-3125 EGM-MV Microvascular Endothelial Cell Growth Includes basal medium and SingleQuots KitKitMedium BulletKitCC-4143 CC-4143 EGM-MV Microvascular Endothelial Cell GrowthKitMedium SingleQuots Supplements and GrowthFactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, and HEPESbuffered saline solution100 ml eachOrdering Information – Other MediaCat. No. NA Cat. No. EU Product Name Product Description Size00190860 00190860 EBM-2 Endothelial Cell Basal Medium-2 1 lCC-3129 CC-3129 EBM Endothelial Cell Basal Medium Phenol red-free 500 ml38North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Microvascular Endothelial Cells and MediaEndothelial cells line the inside surface of blood vessels,heart, lymphatic vessels, body cavities and other organs, ofnormal human tissue.We offer a variety of cell types isolated from microvasculartissue from normal, Type I and Type II diabetic donors.1■■Source––Small vessel endothelial cells are isolated from dermal,lung, cardiac and uterine microvascular tissue■■Applications––Atherosclerosis––Angiogenesis––Arteriosclerosis––Drug uptake or drugdiscovery––Cell-to-cell junctions––Inflammation––Wound healing––Oncology■■Cell Testing and Specifications––Endothelial cells – Test positive for acetylated lowdensity lipoprotein uptake; positive for von WillebrandFactor Expression/Factor VIII; and PECAM-positive forlung microvascular cells. PECAM-positive stain confirmspresence of adherent junctions which control passageof molecules across tissues. Up to 15 populationdoublings are guaranteed <strong>with</strong> normal cells when usingClonetics Media and ReagentsHMVEC-dLyAd stained CD31/Prox-1HMVEC-dAd Hoechst stainHMVEC-LLy von Willebrand Factor and DAPI stainPrimary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com39
Microvascular Endothelial Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaCell Type Description Recommended Media Cryopreserved Cells Proliferating Cells RecommendedSeeding DensityHMVEC-C Cardiac microvascular EGM-2MV BulletKit 3rd passage n/a 2,500–5,000 cells/cm 2Time toSubculture5 to 9 daysHMVEC-L Lung microvascular EGM-2MV BulletKit 3rd or 4th passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysHMVEC-dAd Adult dermal microvascular EGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysHMVEC-dBIAd Adult dermal bloodEGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysmicrovascularHMVEC-dBINeo Neonatal dermal bloodEGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysmicrovascularHMVEC-dLyAd Adult dermal lymphaticEGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 5 to 9 daysmicrovascularHMVEC-dNeo Neonatal dermal microvascular EGM-2MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysHMVEC-dLyNeo Neonatal dermal lymphatic EGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 5 to 9 daysmicrovascularUtMVEC-Myo Uterine microvascular EGM-MV BulletKit 3rd passage 4th or 5th passage 5,000 cells/cm 2 5 to 9 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeDiseased CellsCC-2927 CC-2927 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells –Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type ICC-2928 CC-2928 D-HMVEC – Diseased Cardiac Microvascular Endothelial Cells –Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type IICC-2929 CC-2929 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type ICC-2930 CC-2930 D-HMVEC – Diseased Human Dermal Microvascular Endothelial Cells – Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialDiabetes Type IINormal CellsCC-7016 CC-7016 HMVEC-Bd – Human Bladder Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-7030 CC-7030 HMVEC-C – Human Cardiac Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-2543 CC-2543 HMVEC-dAd – Human Dermal Microvascular Endothelial Cells – Adult Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-2813 CC-2813 HMVEC-dBl-Neo – Human Dermal Blood Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCells – NeonatalCC-2811 CC-2811 HMVEC-dBlAd – Human Dermal Blood Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vial– AdultCC-2810 CC-2810 HMVEC-dLyAd – Human Dermal Lymphatic Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCells – AdultCC-2812 CC-2812 HMVEC-dLyNeo – Human Dermal Lymphatic Microvascular EndothelialCells – NeonatalCryopreserved, in EGM-2MVBulletKit, single donor≥500,000 cells/vialCC-2505 CC-2505 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells,neonatalCC-2516 CC-2516 HMVEC-dNeo – Human Dermal Microvascular Endothelial Cells,neonatalCryopreserved, in EGM-2MVBulletKit, single donorCryopreserved, in EGM-2MVBulletKit, pooled≥500,000 cells/vial≥500,000 cells/vialCC-2527 CC-2527 HMVEC-L – Human Lung Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCC-2564 CC-2564 UtMVEC-Myo – Human Uterine Microvascular Endothelial Cells Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.More ordering information on the next page.40North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Microvascular Endothelial Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3125 CC-3125 EGM-MV Microvascular Endothelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3121 CC-3121 EBM Endothelial Cell Basal Medium 500 mlCC-4143 CC-4143 EGM-MV Microvascular Endothelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, 100 ml eachand HEPES buffered saline solutionSee pages 395–404.Related ProductsEndothelial cells must be cultured in their isolation medium for best results.Nucleofector Kits for Primary Endothelial Cells 202General Ordering and Shipping InformationPage1Primary Cells and Media / Clonetics Human Primary Cells and MediaCryopreserved cells and media products are normally shippedMonday – Thursday for next day delivery. Saturday and Monday deliveriesare available upon special request.For notification of the availability of plated human hepatocytes, contactCustomer Service or your local Sales Representative. .Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com41
Gastrointestinal Cells and Media1Primary Cells and Media / Clonetics Human Primary Cells and MediaThe gastrointestinal tract breaks down food into nutrientsand smaller molecules, which are either absorbed into thebody to provide energy or expelled as a waste. Digestionoccurs mainly in the stomach and small intestine. Smallmolecules are absorbed across the epithelium of the smallintestine and later enter the bloodstream to carry nutrientsto other parts of the body. Intestinal myofibroblasts residesubjacent to the basal membrane in the intestines andmediate molecular flow between the epithelium and cells inthe lamina propria.Our new Cryopreserved InEpC are truly primary cellsrepresenting both villi (enterocytes, goblet, andenteroendocrine cells) and crypts structures.■■Source––Human small intestine, specifically the jejunum■■Applications––Gastrointestinal diseaseor disorder––Oncology––Cell physiology––Drug discovery––Toxicology andcytotoxicityCell Type Description RecommendedMediaHuman intestinal myofibroblasts at second passage stained for α smoothmuscle actin and counterstained <strong>with</strong> DAPI■■Cell Testing and Specifications––Gastrointestinal myofibroblasts – Test ≥90% positivefor α smooth muscle actin and ≤10% positive for theexpression of desmin. Human myofibroblasts areguaranteed through 15 population doublings whenusing Clonetics Media and Reagents.––Intestinal epithelial cells – test ≥90% positive forcytokeratins 8/18 . These cells cannot be subcultured.In combination <strong>with</strong> human intestinal myofibroblasts(InMyoFib), InEpC are able to form very tight cellmonolayer, representing a unique in vitro system tomodel human intestinal homeostasis.CryopreservedCellsProliferating CellsRecommendedSeeding DensityTime toSubcultureH-InMyoFib Intestinal Myofibroblasts SmGM-2 BulletKit 2nd passage 3rd passage 4,000 cells/cm 2 5 to 7 daysInEpi Intestinal Epithelial Cells SmGM-2 BulletKit Primary, - ≥ 50% coverage at 5 to 7 dayscertain cell densityOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2902 CC-2902 H-InMyoFib – Human Intestinal Myofibroblasts Cryopreserved, in SCGM BulletKit ≥500,000 cells/vialCC-2931 CC-2931 InEpC – Human Intestinal Epithelial Cells Cryopreserved, in SmGM-2 BulletKit ≥800,000 cells/vialCC-4540 CC-4540 Human Intestinal Epithelial and Myofibroblast Cell Combo Includes one amp each CC-2902 and CC-2931 ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3182 CC-3182 SmGM-2 Smooth Muscle Growth Medium -2 BulletKit Includes basal medium and SingleQuots Kit KitSee pages 395–404.42North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Lymphatic Cells and MediaEndothelial cells are found in the membrane or monolayerlining of cells taken from lymphatic vessels of heart,lymphatic tissue, surface spinal cord and brain, or anterioreye chamber of normal human tissue.1■■Source––Lymphatic endothelial cells are isolated from dermal orlung microvascular tissue■■Applications––Inflammation––Wound healing––Drug uptake or drugHMVEC-dLyAd stained CD31/Prox-1 HMVEC-LLy von Willebrand Factordiscoveryand DAPI stain––Oncology––Cell-to-cell junctions ■■Cell Testing and Specifications––Endothelial cells – Test positive for acetylated lowdensity lipoprotein uptake; positive for von WillebrandFactor Expression/Factor VIII; PECAM-positive for lungmicrovascular cells which stains for adherent junctionsthat endothelial cells use to control passage ofmolecules across the tissue. Up to 15 populationdoublings guaranteed when using Clonetics Media andReagentsCell Type Description Recommended Media CryopreservedCellsProliferatingCellsRecommendedSeeding DensityTime toSubcultureHMVEC-LLy Lung lymphatic microvascular EGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 4 to 7 daysHMVEC-dLyAd Adult dermal lymphatic microvascular EGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 4 to 7 daysHMVEC-dLyNeo Neonatal dermal lymphaticmicrovascularEGM-2MV BulletKit 3rd passage 4th passage 5,000 cells/cm 2 4 to 7 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2814 CC-2814 HMVEC-Lly – Human Lung Lymphatic Microvascular EndothelialCellsCC-2812 CC-2812 HMVEC-dLyNeo – Human Dermal Lymphatic MicrovascularEndothelial Cells – NeonatalCC-2810 CC-2810 HMVEC-dLyAd – Human Dermal Lymphatic MicrovascularEndothelial Cells – AdultCryopreserved, in EGM-2MV BulletKitCryopreserved, in EGM-2MV BulletKit,single donorCryopreserved, in EGM-2MV BulletKit≥500,000 cells/vial≥500,000 cells/vial≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2Includes basal medium andKitBulletKitSingleQuots KitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2SingleQuots Supplements and Growth FactorsKitSee pages 395–404.Related ProductsPageNucleofector Kits for Primary Endothelial Cells 202Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com43
Mammary Epithelial Cells and Media1Mammary epithelial cells are isolated from glandular tissuein adult human breast tissue. Cells undergo changes inmorphology and function throughout adulthood especiallyduring pregnancy and lactation.Primary Cells and Media / Clonetics Human Primary Cells and Media■■Source––Human adult breast tissue■■Applications––Breast cancer––Cellular function and differentiation––Physiology––Toxicology––Hormone regulation and response■■Cell Testing and Specifications––Human mammary epithelial cells – Test positive forcytokeratins 14 and 18, and negative for cytokeratin 19and are guaranteed through 15 population doublingswhen using Clonetics Media and ReagentsCell Type Description Recommended Media CryopreservedCellsHMEC 95% confluentHMEC stained for cytokeratin 18ProliferatingCellsRecommendedSeeding DensityTime toSubcultureHMEC Mammary Epithelial MEGM BulletKit 6th or 7th passage 7th or 8th passage 2,500 cell/cm 2 6 to 9 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2551 CC-2551 HMEC – Human Mammary Epithelial Cells Cryopreserved, in MEGM BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3150 CC-3150 MEGM Mammary Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3151 CC-3151 MEBM Mammary Epithelial Cell Basal Medium 500 mlCC-3152 CC-3152 MEBM Mammary Epithelial Cell Basal Medium Sodium bicarbonate-free 500 mlCC-3153 CC-3153 MEBM Mammary Epithelial Cell Basal Medium Phenol red-free 500 mlCC-3051 CC-3051 MEGM Complete Mammary Epithelial Cell Growth Medium Includes 1 ea. CC-3051A and CC-4009 500 mlCC-4136 CC-4136 MEGM Mammary Epithelial Cell Growth MediumKitSingleQuots Supplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee pages 395–404.Related ProductsNucleofector Kits for Primary Epithelial Cells 206Page44North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Neural Cells and MediaClonetics Neural Cells are used to study the function of thecentral nervous system and how neural cells interact innormal tissue. Astrocytes are glial cells found in the brainand spinal cord that play a critical role in maintenance,support and repair of nervous tissue.1■■Source––Human brain cortex■■Applications––Neurogenesis research––Pharmacology––Cell physiology––Parkinson’s disease––Injury––Astrocyte-mediated neurotoxicity––Alzheimer’s diseaseCell Type Description RecommendedMediaCryopreservedCellsNHA stained positive for GFAP by indirect immunofluorescence staining■■Cell Testing and Specifications––Normal human astrocytes – Test positive for GFAP andare guaranteed through 10 population doublings whenusing Clonetics Media and ReagentsProliferatingCellsRecommendedSeeding DensityTime toSubcultureNHA Astrocytes AGM BulletKit 1st passage 2nd passage 5,000 cells/cm 2 6 to 8 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2565 CC-2565 NHA – Human Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3186 CC-3186 AGM Astrocyte Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3187 CC-3187 ABM Astrocyte Basal Medium 200 mlCC-4123 CC-4123 AGM Astrocyte Growth Medium SingleQuots SupplementsKitand Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee pages 395–404.Primary Cells and Media / Clonetics Human Primary Cells and MediaRelated ProductsPageRat and Mouse Neural Cells 70Adherent Nucleofection 170Nucleofector Kits for Primary Neural Cells 217Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com45
Ocular Cells and Media1Primary RPE cells are hexagonal cells that are denselypacked <strong>with</strong> pigment granules. They play a critical role invisual function and photoreceptor viability.Primary Cells and Media / Clonetics Human Primary Cells and Media■■Source––Human eye tissue■■Applications––Proliferative retinopathy––Age related macular degeneration––Retinitis pigmentosa––Stargardt’s disease––Blood-retinal barrier research––Toxicology and cytotoxicity––Diabetic retinopathy––BlindnessRPE cells at passage 3 stained fortight junction marker ZO-1 counterstainedDAPIRPE cells at stained for panCytokeratin, counterstained <strong>with</strong>DAPI■■Cell Testing and Specifications––RPE cells – Test ≥90% positive for pancytokeratinmarker, ≤10% positive for fibroblast contamination,≥90% for tight conjunction marker and ≤1% positive forendothelial marker CD31. RPE cells are guaranteedthrough 5 population doublings when using CloneticsMedia and ReagentsCell Type Description Recommended Media Cryopreserved Cells Proliferating Cells Recommended SeedingDensityTime toSubcultureh-RPERetinal pigmentepithelial cellsRtEGM BulletKit 2nd passage 3rd passage 10,000 cells/cm 2 5 to 7 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size00194987 00194987 H-RPE – Human Retinal Pigment Epithelial Cells Cryopreserved, in RtEGM BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description Size00195409 00195409 RtEGM Retinal Pigment Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit Kit00195406 00195406 RtEBM Retinal Epithelial Cell Basal Medium 500 ml00195407 00195407 RtEGM Retinal Epithelial Cell Growth Medium SingleQuotsSupplements and Growth FactorsKitSee pages 395–404.Related ProductsRat Retinal Cells 71Page46North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pancreatic IsletsPancreatic islets are hormone-producing regions inpancreas. These islets consist of beta cells which produceinsulin in the body. Pancreatic islets are being utilized indiabetes research as these islets restore beta-cell functionyielding better regulation of insulin levels.1■■Source––Islets are isolated from endocrine regions of thepancreas■■Applications––Islet Grafting Survival––Immunosuppression––Insulin Production––Cell Metabolism––Diabetes (Type I and Type II); Hypoglycemia■■Cell Testing and Specifications––Islets are tested for IEQ count, sterility, purity andviability prior to shipment. Each batch tests negative forGram Stain, HIV-1, Hep B & C.––Islets are guaranteed to meet >= 70% purity andviability. Additional testing is available upon request(refer to table below).TestOxygen Consumption RateDNA QuantitationGlucose Stimulated Insulin ResponseBeta-cell compositionTurnaround Time1 day1 day4 days14 daysPancreatic isletsPancreatic isletsOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size201981 201981 Fresh Human Pancreatic Islets Fresh, in islets supplemented medium 100,000 islets/flask201983 201983 Fresh Human Pancreatic Islets Fresh, in islets supplemented medium 20,000 islets/flask201984 201984 Fresh Human Pancreatic Islets Fresh, in islets supplemented medium 10,000 islets/flask201985 201985 Fresh Human Pancreatic Islets Fresh, in islets supplemented medium 5,000 islets/flask202998 202998 Fresh Human Pancreatic Islets Fresh, in islets supplemented medium 2,000 islets/flaskFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com47
Prostate Cells and Media1Prostate cells provide a glandular function in the body bygenerating fluid which serves several functions inreproduction.Primary Cells and Media / Clonetics Human Primary Cells and Media■■Source––Prostate epithelial, stromal and smooth muscle tissuedepending on cell type■■Applications––Physiology––Drug discovery––Cancer research––Procreation research■■Cell Testing and Specifications––Prostate epithelial cells – Test positive for cytokeratin(clone 8.13), prostate stromal cells test positive forvimentin and negative for pan cytokeratin. Bothepithelial and stromal cell types are guaranteed through15 population doublings when using Clonetics Mediaand Reagents––Prostate smooth muscle cells – Stain positive forα-actin and are guaranteed through 10 populationdoublings when using Clonetics Media and ReagentsCell Type Description RecommendedMediaPrEC – peroxidase stain for cytokeratin, clone 8.13PrSC stained for vimentinCryopreserved Cells Proliferating Cells RecommendedSeeding DensityTime toSubculturePrEC Prostate epithelial PrEGM BulletKit 1st or 2nd passage 2nd or 3rd passage 2,500 cells/cm 2 6 to 9 daysPrSC Prostate stromal SCGM BulletKit 3rd or 4th passage 4th or 5th passage 3,500 cells/cm 2 6 to 9 daysPrSMC Prostate smooth muscle SmGM-2 BulletKit 2nd or 3rd passage 3rd or 4th passage 3,500 cells/cm 2 6 to 9 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2508 CC-2508 PrSC – Human Prostate Stromal Cells Cryopreserved, in SCGM BulletKit ≥500,000 cells/vialCC-2555 CC-2555 PrEC – Human Prostate Epithelial Cells Cryopreserved, in PrEGM BulletKit ≥500,000 cells/vialCC-2587 CC-2587 PrSMC – Human Prostate Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.More ordering information on the next page.48North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Prostate Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3165 CC-3165 PrEBM Prostate Epithelial Cell Basal Medium Serum-free 500 mlCC-3166 CC-3166 PrEGM Prostate Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4177 CC-4177 PrEGM Prostate Epithelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3205 CC-3205 SCGM Stromal Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3204 CC-3204 SCBM Stromal Cell Basal Medium 500 mlCC-4181 CC-4181 SCGM Stromal Cell Growth Medium SingleQuots Supplements andKitGrowth FactorsCC-4182 CC-4182 HCM Hepatocyte Culture Medium SingleQuots Supplements andKitGrowth FactorsCC-3181 CC-3181 SmBM Smooth Muscle Cell Basal Medium 500 mlCC-4149 CC-4149 SmGM-2 Smooth Muscle Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, 100 ml eachand HEPES buffered saline solutionPrEGM media, see page 400, SCGM media, see page 404.Related ProductsSertoli Cells 57Cell Culture Reagents 138Nucleofector Kits for Primary Epithelial Cells 206Nucleofector Kits for Primary Smooth Muscle Cells 214Page1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com49
Pulmonary Cells and Media1Primary Cells and Media / Clonetics Human Primary Cells and MediaPulmonary cells are found in the lungs and can be usedto study respiration including cilia movement, mucusproduction, gas exchange, air movement, and pulmonaryvascular physiology.We offer these airway cell types from normal, asthma, andCOPD diagnosed donors. We have cells from Cystic Fibrosisdonors when tissue becomes available.■■Source––Human small airway epithelial cells isolated from thedistal portion of the lung in the 1 mm bronchiole area––Human bronchial/tracheal epithelial cells isolated fromthe epithelial cells that line the airway above thebifurcation of the lungs––Small vessel endothelial cells are isolated from lungmicrovascular tissue––Human lung fibroblasts are isolated from adult lungtissue––Human bronchial smooth muscle cells are isolated fromthe major bronchia––Diseased cell types taken from donors that werediagnosed <strong>with</strong> either asthma or COPD. Certaincharacteristics of diseased samples may vary; pleasecontact Scientific Support for further donor informatioN■■Applications––Cystic fibrosis––Respiratory disease––Air /Liquid interface––COPD––Respiratory distress––Oncology––Inhalation technology––Asthma––Basic research––Drug uptake studiessyndrome■■Cell Testing and Specifications––Human bronchial/tracheal epithelial cells and smallairway epithelial cells – Characterized by morphologicalobservation throughout serial passage and SAEC stainpositive for cytokeratin 19, both are guaranteedthrough 15 population doublings when using CloneticsMedia and Reagents––Human lung fibroblasts – Test negative for vonWillebrand Factor Expression/Factor VIII, cytokeratins18 and 19, and smooth muscle α-actin and areguaranteed through 15 population doublings whenusing Clonetics Media and Reagents––Smooth muscle cells – Stain positive for α-actin andnegative for von Willebrand Factor Expression/FactorVIII after differentiation and are guaranteed through 15population doublings when using Clonetics Media andReagentsNHBE – Excellent packed cuboidalmorphologyNHBE – 25 days post air lift grownin B-ALI BulletKit stained forcilia <strong>with</strong> β-tubulinBSMC – Stained for α smoothmuscle actin, counterstained <strong>with</strong>DAPISAEC – Stained for Cytokeratin 19NHBE – Cross section onmembrane, day 26 post air liftgrown in B-ALI BulletKit––Endothelial cells – Test positive for acetylated lowdensity lipoprotein uptake; von Willebrand FactorExpression/Factor VIII; and PECAM-positive for lungmicrovascular cells. PECAM-positive stain confirmspresence of adherent junctions which control passageof molecules across tissues. Up to 15 populationdoublings are guaranteed when using Clonetics Mediaand Reagents; individual cell types may varyOrdering Information on the next page.50North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pulmonary Cells and MediaContinuedCell Type Description Recommended Media CryopreservedCellsSAEC Small airway epithelial SAGM BulletKit 1st or 2ndpassageProliferating Cells2nd or 3rdpassageRecommendedSeeding DensityTime toSubculture2,500 cells/cm 2 5 to 9 daysNHBE <strong>with</strong> RA Bronchial/Tracheal epithelial BEGM BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 daysNHBEBronchial/Tracheal epithelial BEGM BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 days<strong>with</strong>out RADHBE-AsDiseased Bronchial/Tracheal BEGM BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 daysepithelial – AsthmaDHBE-COPD Diseased Bronchial/Tracheal BEGM BulletKit 1st passage 2nd passage 3,500 cells/cm 2 6 to 9 daysepithelial – COPDNHLF Lung fibroblasts FGM-2 BulletKit 2nd passage 3rd passage 2,500 cells/cm 2 6 to 9 daysDHLF-As Diseased Lung fibroblasts – FGM-2 BulletKit 2nd passage 3rd passage 2,500 cells/cm 2 6 to 9 daysAsthmaDHLF-COPD Diseased Lung fibroblasts – FGM-2 BulletKit 2nd passage 3rd passage 2,500 cells/cm 2 6 to 9 daysCOPDHMVEC-L Lung microvascular EGM-2MV BulletKit 3rd or 4th4th or 5th passage 5,000 cells/cm 2 5 to 9 dayspassageHPAEC Pulmonary artery EGM-2 BulletKit 3rd passage 4th passage 2,500–5,000 cells/ 5 to 9 dayscm 2BSMC Bronchial SMC SmGM-2 BulletKit 2nd passage 3rd or 4th passage 3,500 cells/cm 2 6 to 10 daysDBSMC-As Diseased Bronchial SMC – SmGM-2 BulletKit 2nd passage 3rd or 4th passage 3,500 cells/cm 2 6 to 10 daysAsthmaDBSMC-COPD Diseased Bronchial SMC – SmGM-2 BulletKit 2nd passage 3rd or 4th passage 3,500 cells/cm 2 6 to 10 daysCOPDCC-2581Pulmonary Artery SmoothMuscleSmGM-2 BulletKit 3rd passage 4th or 5th passage 3500 cells/cm 2 6 to 10 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeDiseased CellsCC-2932 CC-2932 D-SAEC-As – Diseased Small Airway Epithelial Cells – Asthma Cryopreserved, in SAGM BulletKit ≥500,000 cells/vialCC-2933 CC-2933 D-SAEC-CF – Diseased Small Airway Epithelial Cells – Cystic Fibrosis Cryopreserved, in SAGM BulletKit ≥500,000 cells/vialCC-2934 CC-2934 D-SAEC-COPD – Diseased Small Airway Epithelial Cells – COPD Cryopreserved, in SAGM BulletKit ≥500,000 cells/vial00194850 00194850 D-BSMC-As – Diseased Bronchial Smooth Muscle Cells – Asthma Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vial00194911 00194911 D-HBE-As – Diseased Human Bronchial/Tracheal Epithelial Cells – Cryopreserved, in BEGM BulletKit ≥500,000 cells/vialAsthma00194911S 00194911S D-HABE-As – Diseased Human Bronchial/Tracheal Epithelial Cells – Cryopreserved, in B-ALI BulletKit ≥500,000 cells/vialAsthma for B-ALI Bronchial Air Liquid Interface00194850 00194850 D-BSMC-As – Diseased Bronchial Smooth Muscle Cells – Asthma Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vial00195274 00195274 D-BSMC-COPD – Diseased Bronchial Smooth Muscle Cells – COPD Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vial00195275 00195275 D-HBE-COPD – Diseased Human Bronchial/Tracheal Epithelial Cells Cryopreserved, in BEGM BulletKit ≥500,000 cells/vial– COPD00195275S 00195275S D-HBE-COPD – Diseased Human Bronchial/Tracheal Epithelial Cells – Cryopreserved, in B-ALI BulletKit ≥500,000 cells/vialCOPD for B-ALI Bronchial Air Liquid Interface00195277 00195277 D-HLF-COPD – Diseased Human Lung Fibroblast Cell – COPD Cryopreserved, in FGM-2 BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com51
Pulmonary Cells and MediaContinued1Primary Cells and Media / Clonetics Human Primary Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeNormal CellsCC-2547 CC-2547 SAEC – Human Small Airway Epithelial Cells Cryopreserved, in SAGM BulletKit ≥500,000 cells/vialCC-2547S CC-2547S SAEC – Small Airway Epithelial Cells for S-ALI Air-Liquid-Interface Cryopreserved, in SAGM BulletKit ≥500,000 cells/vialMediumCC-2540S CC-2540S NHBE – Normal Human Bronchial/Tracheal Epithelial Cells for B-ALI Cryopreserved, in BEGM BulletKit, ≥500,000 cells/vialBronchial Air Liquid Interfaceisolated and cultured <strong>with</strong> retinoic acidCC-2541 CC-2541 NHBE – Human Bronchial/Tracheal Epithelial Cells Cryopreserved, in BEGM BulletKit, ≥500,000 cells/vialisolated and cultured <strong>with</strong> retinoic acidCC-2512 CC-2512 NHLF – Normal Human Lung Fibroblasts Cryopreserved, in FGM-2 BulletKit ≥500,000 cells/vialCC-2527 CC-2527 HMVEC-L – Human Lung Microvascular Endothelial Cells Cryopreserved, in EGM-2MV≥500,000 cells/vialBulletKitCC-2530 CC-2530 HPAEC – Human Pulmonary Artery Endothelial Cells Cryopreserved, in EGM-2 BulletKit ≥500,000 cells/vialCC-2581 CC-2581 HPASMC - Human Pulmonary Artery Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2576 CC-2576 BSMC – Human Bronchial Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3118 CC-3118 SAGM Small Airway Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3119 CC-3119 SABM Small Airway Epithelial Cell Basal Medium Serum-free 500 mlCC-4124 CC-4124 SAGM Human Small Airway Epithelial Cell Growth MediumKitSingleQuots Supplements and Growth FactorsCC-3170 CC-3170 BEGM Bronchial Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots KitKit, serum-freeCC-3171 CC-3171 BEBM Bronchial Epithelial Cell Basal Medium 500 mlCC-4175 CC-4175 BEGM Bronchial Epithelial Cell Growth Medium SingleQuotsKitSupplements and Growth Factors00193514 00193514 B-ALI Bronchial Air Liquid Interface Medium BulletKit Includes growth basal medium,Kitdifferentiation basal media and SingleQuotsKit, only sold as BulletKit MediumCC-4539 CC-4539 S-ALI Small Airway Air Liquid Interface Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3132 CC-3132 FGM-2 Fibroblast Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-3131 CC-3131 FBM Fibroblast Basal Medium 500 mlCC-4126 CC-4126 FGM-2 Fibroblast Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3162 CC-3162 EGM-2 Endothelial Cell Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-4176 CC-4176 EGM-2 Endothelial Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 Includes basal medium and SingleQuots Kit KitBulletKitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth FactorsCC-3205 CC-3205 SCGM Stromal Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3204 CC-3204 SCBM Stromal Cell Basal Medium 500 mlCC-4181 CC-4181 SCGM Stromal Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3182 CC-3182 SmGM-2 Smooth Muscle Growth Medium -2 BulletKit Includes basal medium and SingleQuots Kit Kit52North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pulmonary Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3181 CC-3181 SmBM Smooth Muscle Cell Basal Medium 500 mlCC-4149 CC-4149 SmGM-2 Smooth Muscle Cell Growth Medium-2 SingleQuotsKitSupplements and Growth FactorsCC-3124 CC-3124 EGM Endothelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3121 CC-3121 EBM Endothelial Cell Basal Medium 500 mlCC-4133 CC-4133 EGM Endothelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3125 CC-3125 EGM-MV Microvascular Endothelial Cell Growth Medium Includes basal medium and SingleQuots Kit KitBulletKitCC-4143 CC-4143 EGM-MV Microvascular Endothelial Cell Growth Medium KitSingleQuots Supplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution,and HEPES buffered saline solution100 ml eachOrdering Information – Other MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3024 CC-3024 EGMComplete Endothelial Cell Growth Medium With 2% FBS 500 mlCC-3129 CC-3129 EBM Endothelial Cell Basal Medium Phenol red-free 500 mlRelated ProductsSee pages 395–404.Nucleofector Kits for Primary Endothelial and Epithelial Cells 202 and 206Nucleofector Kits for Primary Smooth Muscle Cells 214Page1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com53
Renal Cells and Media1Renal cells are found in the kidneys. They eliminate wasteproducts and modulate electrolytes, pH and blood plasmavolume.Primary Cells and Media / Clonetics Human Primary Cells and Media■■Source––Human kidney tissue layers specific to the designatedcell type; epithelial (a mixture of cortex and glomerular),cortical epithelial (a mixture of RPTEC and distaltubule), proximal tubule epithelial (proximal tubule),and mesangial cells (renal glomerulus and modifiedSMC between capillaries)■■ApplicationsOur renal proximal tubule cells are available fromnormal or Type 2 diabetic donors.––Physiology––Glomerulonephritis––Cancer research––Prostaglandin activity––Cytokine production ––Toxicology––Cellular functiondifferentiation––Phagocytosis ofimmune complexes■■Cell Testing and Specifications––RPTEC – Test positive for γ-GTP––NHMC – Test positive for fibronectin and negative forcytokeratin 19 and von Willebrand Factor/Factor VIII––HRE cells – Stain positive for pan cytokeratin––HRCE – Stain positive for cytokeratin––All cell types – Are guaranteed through 15 populationdoublings when using Clonetics Media and ReagentsCell Type Description Recommended Media CryopreservedCellsRPTEC – Stained positive for g-GTPRPTEC–100% confluencyProliferatingCellsRecommendedSeeding DensityTime toSubcultureRPTEC Proximal tubule REGM BulletKit 1st or 2nd passage 2nd or 3rd passage 2,500 cells/cm 2 5 to 9 daysHRCE Cortical epithelial REGM BulletKit 1st or 2nd passage 2nd or 3rd passage 2,500 cells/cm 2 5 to 9 daysHRE Renal epithelial REGM BulletKit 1st passage 2nd passage 2,500 cells/cm 2 5 to 9 daysNHMC Mesangial cells MsGM BulletKit 3rd passage 4th passage 3,500 cells/cm 2 5 to 9 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2554 CC-2554 HRCE – Human Renal Cortical Epithelial Cells Cryopreserved, in REGM BulletKit ≥500,000 cells/vialCC-2556 CC-2556 HRE – Human Renal Epithelial Cells Cryopreserved, in REGM BulletKit ≥500,000 cells/vialCC-2559 CC-2559 NHMC – Normal Human Mesangial Cells Cryopreserved, in MsGM BulletKit ≥500,000 cells/vialCC-2553 CC-2553 RPTEC – Human Renal Proximal Tubule Epithelial Cells Cryopreserved, in REGM BulletKit ≥500,000 cells/vialCC-2925 CC-2925 D-RPTEC – Diseased Human Renal Proximal TubuleCryopreserved, in REGM BulletKit≥500,000 cells/vialEpithelial Cells – Diabetes Type IIFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.54North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Renal Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-4127 CC-4127 REGM Renal Epithelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3190 CC-3190 REGM Renal Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3191 CC-3191 REBM Renal Epithelial Cell Basal Medium 500 mlCC-4146 CC-4146 MsGM Mesangial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3146 CC-3146 MsGM Mesangial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3147 CC-3147 MsBM Mesangial Cell Basal Medium 500 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee pages 395–404.Related ProductsCell Culture Reagents 138Nucleofector Kits for Primary Epithelial Cells 206Page1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com55
Reproductive Cells and Media1Primary Cells and Media / Clonetics Human Primary Cells and MediaThe human reproductive system is made up of very diverseorgans which work together for the purpose of reproduction.Both male and female reproductive cells are available forthe study of reproductive science and certain genderrelated diseases or disorders.■■Source––Human male and female reproductive systemsincluding prostate, testes, cervical, and uterine tissue■■Applications––Physiology––Cancer research––Toxicology––Toxic Shock Syndrome––Drug discovery––Procreation research––Male infertility––Human papillomavirus■■Cell Testing and Specifications––Prostate epithelial cells – Test positive for cytokeratin(clone 8.13), prostate stromal cells test positive forvimentin and negative for pan cytokeratin. Bothepithelial and stromal cell types are guaranteed through15 population doublings when using Clonetics Mediaand Reagents––Prostate smooth muscle cells – Stain positive forα-actin and are guaranteed to 10 population doublingswhen using Clonetics Media and Reagents––Uterine smooth muscle cells – Stain positive for α-actinand negative for von Willebrand Factor afterdifferentiation––Uterine MV endothelial cells – Stain positive foracetylated low density lipoprotein uptake and for vonWillebrand Factor Expression/Factor VIII, and areguaranteed through 15 population doublings whenusing Clonetics Media and Reagents––Sertoli cells – Test ≥70% positive for GATA-4 and Sox-9by FACS analysisSertoli cell <strong>with</strong> intracellular lipid droplets stained <strong>with</strong> AdipoRed AssayReagentPrEC – Peroxidase stain for cytokeratin, clone 8.13UtSMC – Stained for smooth muscle actinCell Type Description Recommended Media Cryopreserved Cells ProliferatingCellsRecommendedSeeding DensityTime toSubcultureUtMVEC-Myo Uterine microvascular EGM-MV BulletKit 3rd passage 4th or 5th passage 5000 cells/cm 2 5 to 9 daysUtSMC Uterine smooth muscle SmGM-2 BulletKit 3rd passage 4th or 5th passage 3,500 cells/cm 2 6 to 10 daysUASMC Umbilical artery SmGM-2 BulletKit 3rd passage 4th or 5th passage 3,500 cells/cm 2 6 to 10 daysPrEC Prostate epithelial PrEGM BulletKit 1st or 2nd passage 2nd or 3rd passage 2,500 cells/cm 2 6 to 9 daysPrSC Prostate stromal SCGM BulletKit 3rd or 4th passage 4th or 5th passage 3,500 cells/cm 2 6 to 9 daysPrSMC Prostate smooth muscle SmGM-2 BulletKit 2nd or 3rd passage 3rd or 4th passage 3,500 cells/cm 2 6 to 9 daysHSeC Sertoli cells SeGM BulletKit 2nd passage 3rd passage 4,500 cells/cm 2 7 to 10 daysCrEC Cervical Epithelial KGM-Gold BulletKit 2nd passage 3rd passage 3,500 cells/cm 2 5 to 9 days56North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Reproductive Cells and MediaContinuedOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2562 CC-2562 UtSMC – Human Uterine Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2564 CC-2564 UtMVEC-Myo – Human Uterine Microvascular Endothelial Cryopreserved, in EGM-2MV BulletKit ≥500,000 cells/vialCellsCC-2579 CC-2579 UASMC – Human Umbilical Artery Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2587 CC-2587 PrSMC – Human Prostate Smooth Muscle Cells Cryopreserved, in SmGM-2 BulletKit ≥500,000 cells/vialCC-2508 CC-2508 PrSC – Human Prostate Stromal Cells Cryopreserved, in SCGM BulletKit ≥500,000 cells/vialCC-2555 CC-2555 PrEC – Human Prostate Epithelial Cells Cryopreserved, in PrEGM BulletKit ≥500,000 cells/vialMM-HSE-2305 MM-HSE-2305 HSeC – Human Sertoli Cells Cryopreserved, in SeGM BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3156 CC-3156 EBM-2 Endothelial Cell Basal Medium-2 500 mlCC-3202 CC-3202 EGM-2MV Microvascular Endothelial Cell Growth Medium-2 Includes basal medium and SingleQuots Kit KitBulletKitCC-4147 CC-4147 EGM-2MV Microvascular Endothelial Cell Growth Medium-2KitSingleQuots Supplements and Growth Factors00192151 00192151 KBM-Gold Keratinocyte Basal Medium 500 ml00192060 00192060 KGM-Gold Keratinocyte Growth Medium BulletKit Includes growth basal medium,Kitdifferentiation basal media and SingleQuotsKit, only sold as BulletKit Medium00192152 00192152 KGM-Gold Keratinocyte Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3165 CC-3165 PrEBM Prostate Epithelial Cell Basal Medium Serum-free 500 mlCC-3166 CC-3166 PrEGM Prostate Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4177 CC-4177 PrEGM Prostate Epithelial Cell Growth MediumKitSingleQuots Supplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, 100 ml eachand HEPES buffered saline solutionCC-3204 CC-3204 SCBM Stromal Cell Basal Medium 500 mlCC-3205 CC-3205 SCGM Stromal Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4181 CC-4181 SCGM Stromal Cell Growth Medium SingleQuotsKitSupplements and Growth Factors00191051 00191051 SeBM Sertoli Cell Basal Medium 500 ml00191053 00191053 SeGM Sertoli Cell Growth Medium BulletKit Includes growth basal medium,Kitdifferentiation basal media and SingleQuotsKit, only sold as BulletKit Medium00191052 00191052 SeGM Sertoli Cell Growth Medium SingleQuotsSupplements and Growth FactorsKitSee pages 395–404.1Primary Cells and Media / Clonetics Human Primary Cells and MediaRelated ProductsPageNucleofector Kits for Primary Endothelial Cells 202Nucleofector Kits for Primary Epithelial Cells 206Nucleofector Kits for Primary Smooth Muscle Cells 214Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com57
Skeletal and Connective Tissue Cells and Media1Primary Cells and Media / Clonetics Human Primary Cells and MediaSkeletal cells provide primary structural support as bone.Osteoblasts produce bone matrix and prime it formineralization. Chondrocytes produce and maintainextracellular cartilage matrix. Cartilage provides jointcushioning and facilitates joint articulation. Fibroblasts arefound in the stroma of tissue, where they play severalimportant roles, such as manufacturing growth factors andprotein fibers.■■Source––Human osteoblasts are sourced from spongy bonetissue, and human articular chondrocytes are isolatedfrom the knee joint. Fibroblasts are sourced from theperiodontal ligament■■Applications––Physiology––Joint degeneration––Fibrosis––Joint replacement––Bone repair––Bone formation––Bone disease––Osteoporosis■■Cell Testing and Specifications––Human articular chondrocytes – Test positive fortype II collagen and sulfated proteoglycans afterdifferentiation and are guaranteed through 15population doublings when using Clonetics Media andReagents––Human osteoblasts – Test positive for alkalinephosphatase and bone mineralization and areguaranteed through 10 population doublings whenusing Clonetics Media and ReagentsNHAC-kn undifferentiated 100% confluentDay 21 Differentiated NHOst stained <strong>with</strong> OsteoImage Assay Kit––Periodontal ligament fibroblasts – Stain negative forpan cytokeratin and are guaranteed through 10population doublings when using Clonetics Media andReagentsCell Type Description Recommended Media Cryopreserved Cells Proliferating Cells RecommendedSeeding DensityTime toSubcultureNHOst Osteoblasts OGM BulletKit 2nd or 3rd passage 3rd or 4th passage 5,000 cells/cm 2 6 to 9 daysNHAC-knArticular chondrocytes, CDM BulletKit 2nd passage 3rd passage 10,000 cells/cm 2 4 to 9 dayskneeHPdLFPeriodontal ligamentfibroblastsSCGM BulletKit 2nd passage 3rd passage 3,500 cells/cm 2 6 to 9 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2538 CC-2538 NHOst – Normal Human Osteoblasts Cryopreserved, in OGM BulletKit ≥500,000 cells/vialCC-2550 CC-2550 NHAC-kn – Human Articular Chondrocytes – Knee Cryopreserved, in CGM BulletKit ≥750,000 cells/vialCC-7049 CC-7049 HPdLF – Human Periodontal Ligament Fibroblasts Cryopreserved, in SCGM BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.58North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Skeletal and Connective Tissue Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3217 CC-3217 CBM Chondrocyte Basal Medium 500 mlCC-3226 CC-3226 CDM Chondrocyte Differentiation Basal Medium 250 mlCC-3225 CC-3225 CDM Chondrocyte Differentiation Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4408 CC-4408 CDM Chondrocyte Differentiation Medium SingleQuotsKitSupplements and Growth FactorsCC-3216 CC-3216 CGM Chondrocyte Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4409 CC-4409 CGM Chondrocyte Growth Medium SingleQuots SupplementsKitand Growth FactorsCC-3208 CC-3208 OBM Osteoblast Basal Medium 500 mlCC-3207 CC-3207 OGM Osteoblast Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4194 CC-4194 OGM Osteoblast Growth Medium Differentiation SingleQuots To promote differentiationKitSupplements and Growth FactorsCC-4193 CC-4193 OGM Osteoblast Growth Medium SingleQuots SupplementsKitand Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, 100 ml eachand HEPES buffered saline solutionCC-3204 CC-3204 SCBM Stromal Cell Basal Medium 500 mlCC-3205 CC-3205 SCGM Stromal Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4181 CC-4181 SCGM Stromal Cell Growth Medium SingleQuots Supplementsand Growth FactorsKitSee pages 395–404.Ordering Information – ReagentsCat. No. NA Cat. No. EU Product Name Product Description SizeAdditional reagents required to culture chondrocytesCC-3233 CC-3233 Chondrocyte ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, and HEPES 100 mlbuffered saline solutionCC-3234 CC-3234 Sodium Alginate 50 mlCC-3235 CC-3235 Calcium Chloride Solution (CaCl2) 500 mlCC-3236 CC-3236 Sodium Chloride Solution (NaCl) 500 mlCC-3237 CC-3237 Sodium Citrate Solution 100 mlCC-4398 CC-4398 Ascorbic Acid 25.5 mg/ml 0.5 mlRelated ProductsOsteoImage Mineralization Assay 263Rat Osteoblasts 72Human MSCs 87Rat MSCs 90Human Osteoclast Precursors 83Nucleofector Kits for Primary Bone/Cartilage Cells 198Page1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com59
Skeletal Muscle Cells and Media1Skeletal muscle cells form the striated muscles that attachto bones in the skeletal system to control body movement.Skeletal muscle myoblasts are progenitor cells that giverise to muscle cells.Primary Cells and Media / Clonetics Human Primary Cells and Media■■Source––Human skeletal muscle cells are isolated from theupper arm or upper leg, and human skeletal musclemyoblasts are isolated from post-gestational tissue,usually from quadriceps or psoas tissue■■Applications––Our human skeletal muscle myoblasts are availablefrom normal, Type I or Type II donors.––Gene expression––Receptor mediated function––Differentiation––Neuromuscular disease research––Ion transport––Diabetes––Myopathy■■Cell Testing and Specifications––Human skeletal muscle cells – Test positive for desminand are guaranteed through 15 population doublingswhen using Clonetics Media and Reagents––Human skeletal muscle myoblasts – Test positive fordesmin as HSMM myotubes, when differentiated theyform multinucleated myotubes in serum-poor media, orapproaching confluence. Are guaranteed through 10population doublings <strong>with</strong> normal cells when usingClonetics Media and ReagentsCell Type Description RecommendedMediaSkMC stained positive for DesminDifferentiated skeletal muscle (HSMM) culture stained positive forDesminCryopreserved Cells Proliferating Cells RecommendedSeeding DensityTime toSubcultureHSMM Muscle myoblasts SkGM-2 BulletKit 2nd passage 3rd passage 3,500 cells/cm 2 5 to 9 daysSkMC Skeletal muscle SkGM BulletKit 2nd passage 3rd passage 3,500 cells/cm 2 6 to 10 days60North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Skeletal Muscle Cells and MediaContinuedOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeDiseased CellsCC-2900 CC-2900 D-HSMM – Diseased Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit≥500,000 cells/vial– Diabetes Type ICC-2901 CC-2901 D-HSMM – Diseased Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit≥500,000 cells/vial– Diabetes Type IINormal CellsCC-2561 CC-2561 SkMC – Human Skeletal Muscle Cells Cryopreserved, in SkGM-2 BulletKit ≥500,000 cells/vialCC-2580 CC-2580 HSMM – Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit ≥500,000 cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2900 CC-2900 D-HSMM – Diseased Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit≥500,000 cells/vial– Diabetes Type ICC-2901 CC-2901 D-HSMM – Diseased Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit≥500,000 cells/vial– Diabetes Type IICC-2561 CC-2561 SkMC – Human Skeletal Muscle Cells Cryopreserved, in SkGM-2 BulletKit ≥500,000 cells/vialCC-2580 CC-2580 HSMM – Human Skeletal Muscle Myoblasts Cryopreserved, in SkGM-2 BulletKit ≥500,000 cells/vialCC-3160 CC-3160 SkGM Skeletal Muscle Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3161 CC-3161 SkBM Skeletal Muscle Cell Basal Medium 500 mlCC-3244 CC-3244 SkGM-2 Skeletal Muscle Cell Growth Medium-2SingleQuots Supplements and Growth FactorsKitCC-3245 CC-3245 SkGM-2 Skeletal Muscle Cell Growth Medium-2BulletKitRelated ProductsIncludes basal medium and SingleQuots Kit,<strong>with</strong>out L-glutamineCC-3246 CC-3246 SkBM-2 Skeletal Muscle Cell Basal Medium-2 500 mlCC-4139 CC-4139 SkGM Skeletal Muscle Cell Growth Medium SingleQuotsKitSupplements and Growth Factors17-512F BE17-512F Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO4) <strong>with</strong>out calcium or magnesium 500 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee page 404 .Diabetic Human Skeletal Muscle Myoblasts 61KitPage1Primary Cells and Media / Clonetics Human Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com61
Notes1Primary Cells and Media / Clonetics Human Primary Cells and Media62North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Clonetics Animal Primary Cells and Media1Clonetics Animal Primary Cells and MediaIntroduction64Cardiac Cells and Media 65Fibroblasts Cells and Media 67Neural Cells and Media 68Ocular Cells and Media 71Skeletal Cells and Media 72Cell Culture Reagents 73Primary Cells and Media / Clonetics Animal Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com63
Introduction1Primary Cells and Media / Clonetics Animal Primary Cells and MediaClonetics Animal Primary Cells are provided <strong>with</strong> the samequality standards as the Clonetics Human Cell Products. Allcells are perfor mance tested and test negative formycoplasma, bacteria, yeast and fungi. Clonetics Cells areguaranteed to perform as indicated when used <strong>with</strong>Clonetics Cells, Media and Reagents. Immuno and specialstaining protocols, as well as characteristic morphology,are used to characterize the cells and assure they are thedesignated type. A Certificate of Analysis is available foreach cell type and lot.■ ■ General Cell and Media Information––Proliferating cells (select cell types) are offered in thefollowing formats, Flasks (T-25, T-75, T-150, T-225) andMultiwell Plates (6, 12, 24, 48, and 96-wells) contactCustomer Service for order placement––Cell pellets in RNALater® (select cell types) are availableas well <strong>with</strong> 10 million cells/pellet, contact CustomerService for order placement––Clonetics Cells are guaranteed to perform to ourrelease criteria if cultured in our appropriate media––The media systems are offered as BulletKit Products(basal medium and separately packaged growthfactors) to provide the flexibility to manipulate mediacomponents specific to your applicationGeneral Ordering and Shipping InformationCryopreserved cells and media products are normallyshipped Monday – Thursday for next day delivery.Saturday and Monday deliveries are available uponspecial request.Proliferating cell orders must be placed no later thanTuesday at 5:00pm EST for shipment the followingMonday (for delivery on Tuesday). Orders placed afterTuesday will be held until the next production cycle.When you place your order for proliferating cells, pleaseorder the appropriate media. Other cell types may beavailable upon request. Plate orders are an additionalpassage and take an extra week.Cell pellet orders require 7–10 production days. Pleaseplan accordingly.64North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cardiac Cells and MediaCardiac cells are used to study the functions of the vascularsystem and general pathophysiology of the cardiovascularsystem.1■■Source––Rat cardiac myocytes isolated from neonatal SpragueDawley rat hearts (ventricular tissue)––Bovine endothelial cells are isolated from bovine aortas––Rat aortic smooth muscle cells are isolated from theaorta of 150–200 gram adult male Sprague-Dawleyrats■■Applications––Arrhythmia––Heart failure––Angiogenesis––Vascular research––Cardiomyopathy––Preventative cardiology––Artherosclerosis■■Cell Testing and Specifications––Rat cardiac myocytes – Each vial containsapproximately 4 million viable cells at ≥85% purity.When thawed and cultured, you will obtain ≥80%viability, <strong>with</strong> excellent morphology and connectivity,and cells will display beating at 24 hours in culture.Each lot tests positive for functional syncytiumformation and stain positive for actinin. Cell functionguaranteed when using Clonetics Media and Reagents.Primary cardiac myocyte cells need an appropriatesubstrate to adhere and survive – the preferredsubstrate is nitrocelluloseCell Type Description RecommendedMediabAECBovine aortaendothelialRat cardiac myocytes bAEC stained for acetylated LDLRat AoSMC cells at passage 4 stained for α SM actin––Bovine endothelial cells – Stain positive for acetylatedLDL uptake and are observed for key morphologicaltraits from cryopreservation through confluence––Rat aortic smooth muscle cells – Stain ≥95% positivefor α-actin and negative for VE cadherin and areguaranteed through 12 population doublings whenusing Clonetics Media and ReagentsCryopreserved Cells Proliferating Cells Recommended SeedingDensityTime toSubcultureEGM-MV BulletKit 1st passage 2nd passage 2,500–5,000 cells/cm 2 5 to 9 daysR-CM Rat cardiac myocytes RCGM BulletKit 1st or 2nd passage n/a see below* n/aR-ASMRat aortic smooth DMEM:F12 + supplements 2nd passage 3rd passage 5,000 cells/cm 2 5 to 7 daysmusclePrimary Cells and Media / Clonetics Animal Primary Cells and Media*1 ml cell suspension + 9 ml media in 24-well plate (1 ml/well) or 1 ml cell suspension + 4.3 ml media in 96-well plate (200 μl/well)Ordering information on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com65
Cardiac Cells and MediaContinued1Primary Cells and Media / Clonetics Animal Primary Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeBW-6002 BW-6002 bAEC – Bovine Aortic Endothelial Cells Cryopreserved, in EGM-2MV BulletKit, pooled ≥500,000 cells/vialR-ASM-580 R-ASM-580 R-AoSM – Rat Aortic Smooth Muscle Cells Cryopreserved, in DMEM:F12 ≥500,000 cells/vialR-CM-561 R-CM-561 R-CM Rat Cardiac Myocytes Cryopreserved, in RCGM BulletKit ≥4 million cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3124 CC-3124 EGM Endothelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3125 CC-3125 EGM-MV Microvascular Endothelial Cell GrowthIncludes basal medium and SingleQuots KitKitMedium BulletKitCC-3121 CC-3121 EBM Endothelial Cell Basal Medium 500 mlCC-4133 CC-4133 EGM Endothelial Cell Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-4143 CC-4143 EGM-MV Microvascular Endothelial Cell GrowthKitMedium SingleQuots Supplements and Growth FactorsCC-4515 CC-4515 rCGM Rat Cardiac Myocyte Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3275 CC-3275 rCBM Rat Cardiac Myocyte Basal Medium 200 mlCC-4516 CC-4516 RCGM Rat Cardiac Myocyte Growth MediumKitSingleQuots Supplements and Growth FactorsCC-4519 CC-4519 5-Bromo-2‘Deoxyuridine VialBE04-687Q BE04-687Q Dulbecco‘s Modified Eagle Medium:F12 (DMEM:F12) 1:1 mixture <strong>with</strong> 3.151 g/l glucose, <strong>with</strong>1 lUltraGlutamine I, <strong>with</strong>out HEPES14-503F 14-503F Fetal Bovine Serum – Premium, US Origin, HeatHeat inactivated at 56°C for 30 minutes, screened 500 mlInactivatedfor viruses by full compliance 9CFR (113.53) and theabsence of mycoplasma, 3 × 0.1-micron filteredCC-4083 CC-4083 Gentamicin sulfate / Amphotericin (GA-1000) 5 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachRelated ProductsHuman Cardiac Cells 32,38Nucleofector Kits for Primary Cardiac Cells 199Page66North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Fibroblasts Cells and MediaMouse embryonic fibroblasts are often used as a feederlayer to culture ES cells. They provide both a substrate forthe ES cells to grow on and secrete growth factors necessaryfor ES cells to maintain pluripotency.1■■Source––Mouse primary embryonic fibroblasts dissociated fromday 14 and 15 post-coitus CD-1 mouse embryos,expanded and then cryopreserved as frozen primaries.They have not been treated <strong>with</strong> mitomycin-C■■Applications––Embryonic stem cell research––Feeder layer for other cell types■■Cell Testing and Specifications––Mouse primary embryonic fibroblasts – Stain positivefor vimentin expression, are guaranteed for fivepopulation doublings, and display morphologic andgrowth properties equivalent to freshly prepared cellswhen approved media and supplements are usedCell Type Description RecommendedMediaMEFMouse embryonicfibroblastsDMEM high glucosecontaining 10% FBSCryopreservedCellsMEF stained <strong>with</strong> Vimentin at day 3 of second passage post-thaw andcounterstained <strong>with</strong> DAPIProliferating CellsRecommendedSeeding DensityTime toSubculture1st passage 2nd passage 8000 cells/cm 2 5 to 7 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeM-FB-481 M-FB-481 MEF – Mouse Embryonic Fibroblasts Cryopreserved, in DMEM ≥2 million cells/vialFor proliferating cells and cell pellets in RNALater® contact Customer Service for order placement.Primary Cells and Media / Clonetics Animal Primary Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description Size12-604F BE12-604F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong> L-glutamine 500 mlRelated ProductsNucleofector Kits for Mouse Embryonic Fibroblasts 210PageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com67
Neural Cells and Media1Primary Cells and Media / Clonetics Animal Primary Cells and MediaFrozen primary neuronal cells expedite and simplify cellculture research because they can be thawed and culturedon demand to obtain high quality and high yield cultures ofdissociated primary neurons.Shipped overnight to your laboratory, these high quality,cryopreserved, dissociated primary cells represent a costeffective way to do neuronal primary cell culture, eliminatingcostly and time consuming animal care requirements andallowing you to control the experimental/assay timetable.Cryopreserved neuronal cells can be shipped anywhereand used any time.■■Source––Primary rat neurons isolated from rat brain as a nativemix of high quality primary embryonic brain neuronalcells (including glia)––Rat astrocytes are obtained from rat brain, passagedonce, and cryopreserved––Primary mouse neurons and astrocytes are isolatedfrom two different mouse strains, C57 Black and CD1■■Applications––Transfection––Evaluation of electrophysiological properties,neurotransmitters, receptor function––<strong>Research</strong> typical inhibitory or excitatory ion-channels––Receptor signaling research––Intracellular transport studies––Neurotoxicity research■■Cell Testing and Specifications––Rat neurons – Each vial of rat neuronal cells isguaranteed to be mycoplasma and bacteria free.Additional molecular and immunochemical testing(PGP and Tuj) for quality is done following conditionsthat mimic shipping (specific cell types may vary).Prior to cryopreservation, each vial (1 ml) of corticaland striatal neurons contain approximately 4 millionviable cells. Each vial (0.25 ml) of hippocampusneurons contain approximately 1 million viable cells––Rat astrocytes – Each vial (1 ml) containsapproximately 1 million cells. Following confluence, theastrocytes can be harvested for re-plating or storedfrozen for later use. Each vial of astrocytes is guaranteedmycoplasma and bacteria free. Astrocytes are batchtestedfor growth characteristics and morphology(GFAP)Cortical and striatal rat neurons cells were thawed, co-cultured 21 days,and immunofluorescently stained <strong>with</strong> anti- vesicular GABA transporter(vGAT) and anti-dopamine receptor protein (DARPP).Rat cortical neuronal cells were thawed, cultured 14 days, andimmunofluorescently stained <strong>with</strong> anti-PGP 9.5 and anti β-tubulin.––Mouse neurons – Each vial contains approximately4 million cells that are tested for specific neuronalmarkers depending on cell type––Mouse astrocytes – Are a mixed population isolatedfrom the hippocampus, cortex, and striatum of thebrain. These astrocytes are passaged once andcryopreserved68North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Neural Cells and MediaContinued1Immunofluorescence image of cryo preserved rat cortical cells thawedand cultured 21 days stained <strong>with</strong> anti-PGP 9.5 and anti- neurofilament.Cryopreserved mouse cortical neuronal cells were thawed and cultured12 days, then immunofluorescently stained <strong>with</strong> anti-PGP 9.5 andanti-β-tubulin.Cell Type Description RecommendedCryopreserved Cells Proliferating Cells Culture TimeMediaR-Cx Rat brain cortex neurons PNGM BulletKit Immediate n/a 14–21 daysR-Hi Rat brain hippocampus neurons PNGM BulletKit Immediate n/a 14–21 daysR-Cp Rat brain striatum neurons PNGM BulletKit Immediate n/a 14–21 daysR-Drg Rat dorsal root ganglion neurons PNGM BulletKit Immediate n/a 14–21 daysR-eDrg Embryonic rat dorsal root ganglion neurons PNGM BulletKit Immediate n/a 14–21 daysR-Cb Rat cerebellar neurons PNGM-A BulletKit Immediate n/a 14–21 daysR-HTh Rat brain hypothalamic neurons PNGM BulletKit Immediate n/a 14–21 daysR-CxAs Rat brain cortex astrocytes AGM BulletKit Primary passage n/a 14–21 daysR-HiAs Rat brain hippocampus astrocytes AGM BulletKit Primary passage n/a 14–21 daysR-CpAs Rat brain striatum astrocytes AGM BulletKit Primary passage n/a 14–21 daysR-AsM Rat brain Cx-Hi-Cp mix astrocytes AGM BulletKit Primary passage n/a 14–21 daysM-Cx Mouse brain cortex neurons PNGM BulletKit Immediate n/a 14–21 daysM-Cp Mouse brain striatum neurons PNGM BulletKit Immediate n/a 14–21 daysM-Hi Mouse brain hippocampus neurons PNGM BulletKit Immediate n/a 14–21 daysM-AsM Mouse brain mixed astrocytes AGM BulletKit Primary passage n/a 21 + daysOrdering information on the next page.Primary Cells and Media / Clonetics Animal Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com69
Neural Cells and MediaContinued1Primary Cells and Media / Clonetics Animal Primary Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeM-AsM-430 M-ASM-430 M-AsM – Mouse CD1 Brain Mixed Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialM-AsM-330 M-ASM-330 M-AsM – Mouse CD57 Brain Mixed Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialM-Cp-302 M-CP-302 M-Cp – Mouse C57 Brain Striatum Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialM-Cp-402 M-CP-402 M-Cp – Mouse CD1 Brain Striatum Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialM-Cx-300 M-CX-300 M-Cx – Mouse C57 Brain Cortex Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialM-Cx-400 M-CX-400 M-Cx – Mouse CD1 Brain Cortex Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialM-Hi-401 M-HI-401 M-Hi – Mouse Brain Hippocampus Neurons Cryopreserved, in PNGM BulletKit ≥1 million cells/vialR-AsM-530 R-ASM-530 R-AsM – Rat Brain Cx-Hi-Cp Mix Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialR-CB-503 R-CB-503 R-Cb – Rat Cerebellar Neurons Cryopreserved, in PNGM-A, granule cells ≥4 million cells/vialR-Cp-502 R-CP-502 R-Cp – Rat Brain Striatum Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialR-CpAs-522 R-CPAS-522 R-CpAs – Rat Brain Striatum Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialR-Cx-500 R-CX-500 R-Cx – Rat Brain Cortex Neurons Cryopreserved, in PNGM BulletKit ≥4 million cells/vialR-CxAs-520 R-CXAS-520 R-Cx-As – Rat Brain Cortex Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialR-Drg-505 R-DRG-505 R-DRG – Rat Dorsal Root Ganglion Neurons Cryopreserved, in PNGM BulletKit ≥200,000 cells/vialR-eDRG-515 R-eDRG-515 R-eDRG – Rat Dorsal Root Ganglion Neurons – Embryonic Cryopreserved, in PNGM BulletKit ≥1 million cells/vialR-G-535 R-G-535 R-G – Rat Microglia Cryopreserved, in DMEM ≥2 million cells/vialR-Hi-501 R-HI-501 R-Hi – Rat Brain Hippocampus Neurons Cryopreserved, in PNGM BulletKit ≥1 million cells/vialR-HiAs-521 R-HIAS-521 R-HiAs – Rat Brain Hippocampus Astrocytes Cryopreserved, in AGM BulletKit ≥1 million cells/vialR-HTh-507 R-HTh-507 R-Hth – Rat Brain Hypothalamus Neurons Cryopreserved, in PNGM BulletKit ≥2 million cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-4461 CC-4461 PNGM Primary Neuron Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3256 CC-3256 PNBM Primary Neuron Basal Medium 200 mlCC-4462 CC-4462 PNGM Primary Neuron Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-4511 CC-4511 PNGM-A Primary Neuron Growth Medium – AdultKitSingleQuots Supplements and Growth FactorsCC-4512 CC-4512 PNGM-A Primary Neuron Growth Medium – AdultIncludes basal medium and SingleQuots Kit KitBulletKitCC-3186 CC-3186 AGM Astrocyte Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3187 CC-3187 ABM Astrocyte Basal Medium 200 mlCC-4123 CC-4123 AGM Astrocyte Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee pages 395–404s .Related ProductsPageHuman Neural Cells 45Human Neural Progenitor Cells 82Rat Retinal Cells 71Adherent Nucleofection 170Nucleofector Kits for Primary Neural Cells 21770North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Ocular Cells and MediaThe vertebrate retina is a light sensitive tissue lining theinner surface of the eye. Light strikes the retina, creates animage and initiates a cascade of chemical and electricalevents that ultimately trigger nerve impulses. Theseimpulses are sent to visual centers of the brain through thefibers of the optic nerve.■■Source––Rat retinal cells isolated from neonatal (day 3–4)Sprague-Dawley rats and comprised of the seven celltypes normally found in retina. They are prepared bydissection/dissociation <strong>with</strong>out purification, cryopreserved,and are ready for immediate culture■■Applications––General ophthalmicresearch––Posterior segmentdisease––Neoplasms––Cell therapies––Toxicology andcytotoxicity––Inflammation––Drug delivery––Degeneration––Gene expressionCell Type Description RecommendedMediaRat retinal cells stained for neuron specific class III β-tubulin (Tuj-1) andneuronal protein gene product (PGP 9.5)■■Cell Testing and Specifications––Rat retinal cells – Each lot tests negative formycoplasma and sterility. Immunostaining for neuronspecific class III β-tubulin (Tuj-1), specific neuronalprotein gene product (PGP 9.5), ganglion cell marker,Thy1.1, and GFAPCryopreservedCellsProliferatingCellsCulture TimeR-Ret Rat retinal cells PNGM BulletKit Immediate n/a 14–21 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeR-Ret-508 R-Ret-508 R-Ret-Neo – Rat Retinal Cells, neonatal Cryopreserved, in PNGM BulletKit ≥200,000 cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-4461 CC-4461 PNGM Primary Neuron Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4462 CC-4462 PNGM Primary Neuron Growth Medium SingleQuotsKitSupplements and Growth FactorsCC-3256 CC-3256 PNBM Primary Neuron Basal Medium 200 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, andHEPES buffered saline solution100 ml eachSee pages 395–404 .1Primary Cells and Media / Clonetics Animal Primary Cells and MediaRelated ProductsPageHuman Neural Cells 45Human Neural Progenitor Cells 82Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com71
Skeletal Cells and Media1Primary Cells and Media / Clonetics Animal Primary Cells and MediaSkeletal cells provide primary structural support as bone.Osteoblasts produce bone matrix and prime it formineralization. Bone cells are responsible for the body’sresponse trauma and fracture to strengthen, develop, heal,and grow bone.■■Source––Rat calvariae osteoblasts dissociated from Sprague-Dawley rat embryos (E20, E21)■■Applications––Physiology––Bone formation––Joint replacement––Bone repair––Bone disease––Osteoporosis■■Cell Testing and Specifications––Rat osteoblasts – Are cryopreserved at dissection andeach vial of osteoblasts contains ≥0.35 million viablecells. This will seed into approximately three 6-wellplates for mineralization studies, three T-25 flasks orone T-75 flask for proliferation studies using therecommended plating densities and mediumCell Type Description RecommendedMediaR-OSTRat calvariaeosteoblastsDMEM high glucose and RatMSCGM SingleQuots KitCryopreservedCellsRat osteoblasts stained <strong>with</strong> OsteoImage Assay for mineralization atday 24––Rat osteoblasts – Will undergo at least 12 populationdoublings and are tested for mineralization afterdifferentiation. For mineralization studies, it isrecommended to plate cells directly out ofcyropreservation into multi-well plates. Upon inducingdifferentiation, cells require 3 to 5 weeks to sufficientlyform mineralized nodulesProliferating CellsRecommended SeedingDensityTime toSubculture1st Passage n/a 5,000–7,000 cells/cm 2 5 to 7 daysOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeR-OST-583 R-OST-583 R-OST – Rat Calvariae Osteoblasts Cryopreserved, in DMEM ≥500,000 cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description Size12-604F BE12-604F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong> L-glutamine 500 ml00192820 00192820 Rat MSCGM Mesenchymal Stem Cell Growth MediumKitSingleQuots Supplements and Growth Factors00192829 00192829 Rat Mesenchymal Stem Cell Osteogenic SingleQuotsSupplements and Growth FactorsKitRelated ProductsPageHuman Skeletal and Connective Tissue 58Rat MSCs 90Human MSCs 87OsteoImage Mineralization Assay 26372North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Culture ReagentsOrdering Information – ReagentsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-5002 CC-5002 Trypsin Neutralizing Solution 100 mlCC-5012 CC-5012 Trypsin/EDTA Solution 100 mlCC-5022 CC-5022 HEPES Buffered Saline Solution 100 mlCC-5024 CC-5024 HEPES Buffered Saline Solution 500 mlCC-5034 CC-5034 ReagentPack Subculture Reagents Trypsin/EDTA, trypsin neutralizing solution, and HEPES 100 ml eachbuffered saline solutionT100ARetronectin® Recombinant Human FibronectinFragmentRecombinant human bronectin fragment CH-296 producedin E.coli. When coated on the surface of asks and plates,Retronectin® significantly enhances retrovirus-mediatedgene transfer into mammalian cells.0.5 mgT100BRetronectin® Recombinant Human FibronectinFragmentRecombinant human bronectin fragment CH-296 producedin E.coli. When coated on the surface of asks and plates,Retronectin® significantly enhances retrovirus-mediatedgene transfer into mammalian cells.T110A Retronectin Dish 10 dishes (35mm)Additional Cell Culture Reagents can be found on pages 398–404.Ordering Information – Growth FactorsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-4009 CC-4009 Bovine Pituitary Extract 13 mg/ml 2 mlCC-4068 CC-4068 hFGF – Human Fibroblastic Growth Factor 1 µg/ml 1 mlCC-4098 CC-4098 Bovine Brain Extract 9 mg/ml 5 mlCC-4107 CC-4107 hEGF Human Epidermal Growth Factor 3 µg/ml 0.5 mlCC-4202 CC-4202 Calcium Chloride 300 mM 2 mlCC-4205 CC-4205 Human Transferrin 10 mg/ml 0.5 mlCC-4323 CC-4323 NSF-1 Neural Survival Factor-1 4 mlCC-4398 CC-4398 Ascorbic Acid 25.5 mg/ml 0.5 ml2.5 mgAdditional Cell Culture Reagents can be found on pages 398–4041Primary Cells and Media / Clonetics Animal Primary Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com73
1Primary Cells and Media / Clonetics Animal Primary Cells and Media74North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Poietics Stem Cells and MediaWe do the isolation, you do the research1Poietics Stem Cells and MediaIntroduction76Human Adipose-derived Stem Cells 77Human Bone Marrow Products 78Quadratox 4 Species Panel of Bone MarrowMononuclear Cells 78Mononuclear Cells 79Human CD34 + and CD133 + Progenitor Cells 80Human CD34 + Differentiation Supplements 81Human Neural Progenitor Cells and Media 82Human Osteoclast Precursor Cells and Media 83Human Pluripotent Stem Cells 84Human Preadipocyte Cells and Media 86Human Mesenchymal Stem Cells and Media 88Pluripotent Stem Cell Services 90Rat Mesenchymal Stem Cells and Media 91Primary Cells and Media / Poietics Stem Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com75
Introduction1Primary Cells and Media / Poietics Stem Cells and MediaPoietics Stem Cells, Progenitor Cells, and Media include acollection of human and animal cells derived fromhematopoietic, adipose, and neural systems. Fresh orcryopreserved cells are available from bone marrow, cordblood, and fetal liver. Media systems are available for eachcell type. All cells are obtained through Institutional ReviewBoard-approved donor programs.We also offer a number of highly purified standard cell typesas well as custom cell isolation services. All products arequality controlled and are provided <strong>with</strong> a certificate ofanalysis.General Ordering and Shipping InformationDifferentiating cells are generally available in both freshand cryopreserved formats. Orders for fresh cells areusually scheduled 1–2 weeks ahead of your anticipateduse date. Fresh cells are processed on the same day asdrawn and shipped for overnight delivery (US only).Fresh and cryopreserved cells are shipped Mondaythrough Thursday. Fresh cells are shipped in HBSS <strong>with</strong>5 mM EDTA and 0.5% BSA.76North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Adipose-derived Stem Cells (ADSC)Our human ADSCs are isolated from adipose tissue fromnormal, or Type I and II Diabetic donors. Cells can be selectedfrom lots based on donor characteristics such as age, sex,race, and BMI.The cells are cryopreserved at primary passage and havebeen reported in multiple publications to differentiate downvarious lineages including chondrogenic, osteogenic,adipogenic, myogenic, neural, and endothelial. ADSC GrowthMedium BulletKit has been optimized for cell maintenanceand expansion.■■Applications––Toxicology/drug screening––Regenerative medicine/cell therapy––Obesity––Osteoporosis––Cardiovascular disease■■Specifications––≥1 million viable cells after thaw; >95% pure––Positive for CD13, CD29, CD44, CD73, CD90, CD105,CD166, surface markers––Negative for CD14, CD31, CD45, surface markers––Negative for HIV-1, Hepatitis B and C––Guaranteed to expand through five passagesADSC – derived adipocytes stained<strong>with</strong> Oil Red OADSC – derived osteoblasts stained<strong>with</strong> Alizarin RedADSC – derived chondrocytesstained <strong>with</strong> Alcian BlueOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizePT-5006 PT-5006 HADSC – Human Adipose-Derived Stem Cells Cryopreserved ≥1 million cells/vialPT-5007 PT-5007 D-HADSC – Diseased Human Adipose-derived Stem Cells – Diabetes Type I Cryopreserved ≥1 million cells/vialPT-5008 PT-5008 D-HADSC – Diseased Human Adipose-derived Stem Cells – Diabetes Type II Cryopreserved ≥1 million cells/vial1Primary Cells and Media / Poietics Stem Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-4505 PT-4505 ADSC – Apidose-Derived Stem Cells Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitPT-3273 PT-3273 ADSC – Apidose-Derived Stem Cells Basal Medium 500 mlPT-4503 PT-4503 ADSC – Apidose-Derived Stem Cells Growth MediumSingleQuots Supplements and Growth FactorsFrozen supplementsKitRelated ProductsPageOsteoImage Mineralization Assay 263PGM-2 BulletKit 86Human Mesenchymal Stem Cells 87Nucleofector Kits for Primary Stem Cells 222Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com77
Human Bone Marrow Products1Primary Cells and Media / Poietics Stem Cells and MediaUnprocessed Bone MarrowFresh, unprocessed bone marrow is collected Mondaythrough Thursday. Orders are shipped the same day as thebone marrow is drawn <strong>with</strong> overnight delivery.Our high quality human bone marrow is guaranteed tocontain a minimum of 15 million leukocytes per milliliter(ml).Please contact your Scientific Support Specialist forfurther details on ordering fresh marrow.Quadratox 4 Species Panel of Bone Marrow Mononuclear CellsHuman Stromal CellsWhen bone marrow cells are put into culture under specificconditions, a stromal layer of adherent cells develops over afew weeks. The stromal layer is composed of many celltypes, including fibroblasts, mesenchymal stem cells,adipocytes, endothelial cells and macrophages. Thesestromal cells function as a feeder layer for hematopoieticprogenitors, allowing proliferation and differentiation ofprogenitors to continue for weeks in these cultures <strong>with</strong> noaddition of exogenous cytokines. This long-term culturesystem allows researchers to study an in vitro model of thebone marrow microenvironment, including cell-cellinteractions, adhesion molecules and cytokine secretion.These and many other factors allow for the tight regulationof blood cell production, progenitor cell commitment anddifferentiation, and stem cell renewal.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size1M-125 1M-125 Unprocessed Human Bone Marrow Fresh 25 mlOrder 4 × 1M-125 Unprocessed Human Bone Marrow (fresh, same donor) 100 ml2M-302 2M-302 Human Bone Marrow Stromal Cells Cryopreserved, non-irradiated ≥5 million cells/vialThe Quadratox 4 Species Panel is a panel of bone marrowderivedmono nuclear cells from 4 different mammalianspecies including inbred C57BL/6 mice, purebred beagles,baboons, and humans. Animals and tissues are obtainedfrom AAALAC-accredited facilities.Quadratox Panels can be used to assess the relativetoxicities of chemotherapeutic drugs in pre-clinicalresearch. Inter-species differences in the relative toxicitiesof numerous chemo therapeutic drugs are well documented.In vitro toxicity data have been shown to contribute to boththe structuring and subsequent interpretation of clinicaltrials. Quadratox Panels may be used for selection of themost appropriate animal models for both efficacy andtoxicology studies and prediction of in vivo humanmyelotoxicity.The Quadratox 4 Species Panel is available as cryo preservedvials of ≥5 million cells/species. Bone marrow mononuclearcells from human, murine, canine, and baboon are alsoavailable individually.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size2S-101 2S-101 Quadratox 4 Species Panel Cryopreserved ≥5 million cells/species2S-101D 2S-101D Human Bone Marrow Mononuclear Cells Cryopreserved ≥5 million cells/vial2S-101C 2S-101C Murine Bone Marrow Mononuclear Cells Cryopreserved ≥5 million cells/vial2S-101B 2S-101B Canine Bone Marrow Mononuclear Cells Cryopreserved ≥5 million cells/vial2S-101A 2S-101A Baboon Bone Marrow Mononuclear Cells Cryopreserved ≥5 million cells/vial78North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Mononuclear CellsHuman Bone Marrow Mononuclear CellsBone marrow is the primary source of hematopoieticprogenitors. These progenitors can be readily isolated fromthe mononuclear cell fraction. Bone marrow mononuclearcells are available from a large and varied donor pool.Both bone marrow and cord blood mononuclear cells areprepared by centrifugation in a density cell separationmedium (Ficoll-Paque®, GE HealthCare Bio-Sciences AB).Isolating the mononuclear cells eliminates erythrocytesand granulocytes, producing a more stable cell product.Mononuclear cells can be used directly in hematopoieticassays, or as the starting material for isolating CD34+progenitor cells. Cells are available fresh or cryopreserved.When shipped fresh, cells are suspended in HBSS containing0.5% BSA and 5 mM EDTA.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size1M-125A 1M-125A Human Bone Marrow Mononuclear Cells Fresh ≥200 million cells/vial1M-125C 1M-125C Human Bone Marrow Mononuclear Cells Fresh ≥25 million cells/vial1M-125D 1M-125D Human Bone Marrow Mononuclear Cells Fresh ≥100 million cells/vial1M-125E 1M-125E Human Bone Marrow Mononuclear Cells Fresh ≥300 million cells/vial2M-125A 2M-125A Human Bone Marrow Mononuclear Cells Cryopreserved ≥200 million cells/vial2M-125B 2M-125B Human Bone Marrow Mononuclear Cell Panel Cryopreserved, 5 donors ≥25 million cells/donor2M-125C 2M-125C Human Bone Marrow Mononuclear Cells Cryopreserved ≥25 million cells/vial2M-125D 2M-125D Human Bone Marrow Mononuclear Cells Cryopreserved ≥100 million cells/vial1Primary Cells and Media / Poietics Stem Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com79
Human CD34 + and CD133 + Progenitor Cells1Primary Cells and Media / Poietics Stem Cells and MediaCD34+ progenitor cells are available from bone marrow,cord blood and fetal liver. The cells are isolated frommononuclear cells using positive immunomagneticselection. Cell purity via FACS is >90%. CD34 is a knownmarker for hematopoietic progenitor cells. As these cellsmature and differentiate, CD34 expression is lost.CD133+ are subset of CD34+ cells. CD133+ progenitor cellsare isolated by positive selection. Cell purity via FACS is≥90%.Fresh cells need to be scheduled 2–4 weeks in advancedepending on the source and volume requirements.RyErythropoiesis from CD34 + Progenitor Cells2.0E+050.0E+0060 500 10,000Erythropoietin (mUnits/ml)25ng/ml SCF10ng/ml SCFOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size1M-101 1M-101 Human Bone Marrow CD34 + Progenitor Cells Fresh ≥100,000 cells/vial1M-101A 1M-101A Human Bone Marrow CD34 + Progenitor Cells Fresh ≥300,000 cells/vial1M-101B 1M-101B Human Bone Marrow CD34 + Progenitor Cells Fresh ≥500,000 cells/vial1M-101C 1M-101C Human Bone Marrow CD34 + Progenitor Cells Fresh ≥1 million cells/vial2M-101 2M-101 Human Bone Marrow CD34 + Progenitor Cells Cryopreserved ≥100,000 cells/vial2M-101A 2M-101A Human Bone Marrow CD34 + Progenitor Cells Cryopreserved ≥300,000 cells/vial2M-101B 2M-101B Human Bone Marrow CD34 + Progenitor Cells Cryopreserved ≥500,000 cells/vial2M-101C 2M-101C Human Bone Marrow CD34 + Progenitor Cells Cryopreserved, volume discount available ≥1 million cells/vial2M-101D 2M-101D Human Bone Marrow CD34 + Progenitor Cells Cryopreserved ≥2 million cells/vial2M-101F 2M-101F Human Bone Marrow CD34 + Progenitor Cells Cryopreserved ≥10 million cells/vial2C-102 2C-102 Human Cord Blood CD133 + Progenitor Cells Cryopreserved ≥500,000 cells/vial2C-102A 2C-102A Human Cord Blood CD133 + Progenitor Cells Cryopreserved ≥100,000 cells/vial2C-101 2C-101 Human Cord Blood CD34 + Progenitor Cells Cryopreserved ≥1 million cells/vial2C-101A 2C-101A Human Cord Blood CD34 + Progenitor Cells Cryopreserved ≥500,000 cells/vial2C-101B 2C-101B Human Cord Blood CD34 + Progenitor Cells Cryopreserved ≥100,000 cells/vial80North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human CD34 + MediaHematopoietic Progenitor Growth Medium (HPGM) is aserum-free medium containing only human proteins thatsupports the proliferation of hematopoietic progenitor <strong>with</strong>the addition of cytokines.1Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-3926 PT-3926 HPGM Hematopoietic Progenitor Growth Medium 500 mlHuman CD34 + Differentiation SupplementsPrimary human bone marrow hematopoietic progenitorsare multipotential cells that can differentiate into severallineages (e.g., myeloid, erythroid and megakaryocyticcells). The myeloid, erythroid and megakaryocyticEasyDiff Supplements allow differentiation of CD34+ cellsinto 3 different hematopoietic lineages in 96-well plates.These EasyDiff Supplements can be used for both drugdiscovery and toxicology. Bone marrow progenitors areextremely sensitive to the toxic side effects of many drugs.Early discovery and elimination of toxic compounds is animportant component of the drug discovery process.Studies have shown that the inhibitory activity of severaldrugs, as measured in in vitro assays of hematopoieticprogenitor differentiation, correlate <strong>with</strong> their in vivoactivities in various animal models (Volpe, 1996;Parchment, 1194). Toxic effects of drugs can also belineage-specific (Scheding, 1994; Silverstein, 1988).Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-4514 PT-4514 EasyDiff Supplement – Erythroid for 96-well platePT-4515 PT-4515 EasyDiff Supplement – Erythroid for 3 × 96-well platesPT-4512 PT-4512 EasyDiff Supplement – Megakaryocyte for 96-well platePT-4513 PT-4513 EasyDiff Supplement – Megakaryocyte for 3 × 96-well platesPT-4510 PT-4510 EasyDiff Supplement – Myeloid for 96-well platePT-4511 PT-4511 EasyDiff Supplement – Myeloid for 3 × 96-well platesPrimary Cells and Media / Poietics Stem Cells and MediaRelated ProductsPageHuman CD34 + Progenitor Cells 80Human Mononuclear Cells 79Human CD34 + Cell Nucleofector Kit 222LGM-3 Lymphocyte Growth Medium 93-95Iscove‘s Modified Dulbecco‘s Medium (IMDM) 106Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com81
Human Neural Progenitor Cells and Media (NHNP)1Primary Cells and Media / Poietics Stem Cells and MediaPoietics Neural Progenitor Cells are cryopreserved asneurospheres isolated from human brain cortex andcryopreserved. We offer 2 optimized media kits speciallyformulated to support the maintenance, and differentiationof the NHNP cells.NPMM Neural Progenitor Maintenance Medium BulletKitcontains the necessary supplements and media for optimalNHNP cell growth and differentiation. This kit includes:––CC-3210 – Neural Progenitor Basal Medium––CC-4241 – Neural Progenitor Maintenance MediumSingleQuots Kit (contains hEGF and hFGF)––CC-4242 – Neural Progenitor Supplement SingleQuotsKit (contains NSF-1 and GA)NPDM Neural Progenitor Differentiation Medium BulletKitcontains the necessary supplements and media for optimalNHNP differentiation. The medium can be customized bysupplementation <strong>with</strong> differentiation-promoting agentssuch as brain-derived neurotrophic factor. This kit includes:––CC-3210 – Neural Progenitor Basal Medium––CC-4242 – Neural Progenitor Supplement SingleQuotsKit■■Benefits––Cryopreserved in primary passage––Guaranteed marker expression when plated ontolaminin––Media to support maintenance and differentiationNHNP stained for -tubulin III and GFAP■■Applications––Drug development––Neurotoxicity––Neurogenesis and CNS function––Neurotransmitter disorders––Electrophysiology■■Cell Testing and Specifications––Cells and media tested together for optimal performance––Cells negative for HIV-1, Hepatitis B and C––Cells negative for mycoplasma, bacteria, yeast andfungi––Cells test positive for β-tublin III and GFAP1. Sold under license from StemCells, Inc. US patents 5,968,829 and5,851, 832.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizePT-2599 PT-2599 NHNP – Human Neural Progenitor Cells Cryopreserved ≥1.2 million cells/vialFor cell pellets in RNALater contact Customer Service for order placement..Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3209 CC-3209 NPMM Neural Progenitor Maintenance Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-3210 CC-3210 NPBM Neural Progenitor Basal Medium 200 mlCC-3229 CC-3229 NPDM Neural Progenitor Differentiation Medium BulletKit Includes basal medium and SingleQuots Kit KitCC-4241 CC-4241 NPMM Neural Progenitor Maintenance MediumKitSingleQuots Supplements and Growth FactorsCC-4242 CC-4242 Neural Progenitor Supplement SingleQuots Supplementsand Growth FactorsKitSee pages 411–433.82North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Osteoclast Precursor Cells (OCP) and Media■■Poietics Osteoclast Precursor Cells and Media Contain:––Cryopreserved human osteoclast precursors––OCP Osteoclast Precursor Medium BulletKit1Osteoclasts are large, multinucleated cells that play anactive role in bone resorption. This cell system has beendesigned for use in high-throughput applications to conductresearch on osteoporosis, bone resorption, and other bonerelateddiseases.OCP Osteoclast Precursor Medium BulletKit includes thebasal medium and supplements needed to induce theosteoclast precursors to differentiate into matureosteoclasts; these differentiated osteoclasts stain positivefor TRAP and express the calcitonin receptor.Poietics Cells, Media, and Reagents are quality testedtogether and guaranteed to give optimum performance as acomplete cell system.Differentiated human osteoclastsresorbing bone fragmentEffects of Calcitonin on Osteoclast-mediated BoneMatrix Degradation in vitroOsteoLyse (RFU)250,000200,000150,000100,00050,000Pits formed from bone resorptionactivity of differentiatedosteoclasts0Control Day 5-6Day 0-6Calcitonin (1nm) ExposureInhibition of bone matrix resorption by calcitonin. Poietics PrimaryHuman Osteoclast Precursors were cultured in differentiation mediumcontaining no calcitonin, calcitonin added only at day 5 and calcitoninadded on days 0 and 5 and assayed after a total of 6 days. Calcitonin,added at day 0, resulted in the osteoclasts becoming refractory tocalcitonin added on day 5.Primary Cells and Media / Poietics Stem Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size2T-110 2T-110 hOPC – Human Osteoclast Precursor Cells Cryopreserved, in OCP BulletKit ≥1 million cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-8001 PT-8001 OCP – Osteoclast Precursor Medium BulletKit Includes basal medium and SingleQuots Kit KitPT-8201 PT-8201 OCP – Osteoclast Precursor Basal Medium 100 mlPT-9501 PT-9501 OCP – Osteoclast Precursor Medium SingleQuotsKitSupplements and Growth FactorsFor related assays using these cells, see pages 257 –258..Related ProductsPageOsteoAssay Human Bone Plate 261OsteoImage Mineralization Assay 263OsteoLyse Assay Kit 262Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com83
Human Pluripotent Stem Cells1Primary Cells and Media / Poietics Stem Cells and MediaHuman Pluripotent Stem Cell-derived Cell TypesLonza and its collaborators utilize proprietary methods forscalable production of physiologically relevant cell typesfrom pluripotent stem cells. These highly pure cellpopulations specifically address issues of limited tissueavailability, donor and preparation variability, and improvethe predictive strength of current animal and cell basedmodels.■■Benefits––Pure human cells; no species extrapolation necessary––Available in large quantities––Avoid difficult isolations, poor yields, low purity and / orlengthy differentiation processes––Minimal lot-to-lot variation––Ready-to-use cryopreserved vials for more flexibility inyour experimentationMotorPlate Human ESC Motor Neuron ProgenitorsLive, functional, adherent hESC-derived motor neuronprogenitors (MNPs)––MotorPlateStandard MNPs – MNPs at approximatelyday 30 after induction of neural differentiation.––MotorPlateMature MNPs – MNPs at approximately day50 after induction of neural differentiation.■■Applications––Cryopreserved MNPs – MNPs frozen down atapproximately day 28 after induction of neuraldifferentiation.––Viability, toxicity, and neuronal outgrowth assays––Maturation studies and functional testing––Motor neuron disease research (ALS, SMA, etc.)■■Cell Testing and Specifications––75% Neuronal Class III β-tubulin (TUJ1)––30% Neurofilament (SMI-32) – mature only––15% of Glial Fibrillary Acid Protein (GFAP)Standard MotorPlate Cells stained for Neuronal Class III β-tubulin (green)and Glial Fibrillary Acid Protein (red). Nuclei are counterstained <strong>with</strong> DAPI(blue).Cryopreserved Human Motor Neuron ProgenitorsIn an effort to better meet the needs of the researchcommunity, we have expanded our MotorPlate product lineto include cryopreserved vials of these cells.■■Benefits––Flexibility – ability to schedule experiments andaccommodate last minute changes––Versatility – utility in more types of experiments––Availability – in stock, no production lead times––Efficiency - Avoid poor yields, low purity and lengthydifferentiation process■■Cell Testing and Specifications––4 million cells per vial––75% Neuronal Class III s-tubulin (TUJ1)––15% of Glial Fibrillary Acid Protein (GFAP)MotorBlast Motor Neuron Culture MediumMotorBlast is designed and optimized for maintenanceand maturation of cryopreserved MNPs derived from humanESCs. NeuroBlast Medium is required for plating ofcryopreserved MNPs.––Poly-D-lysine <strong>with</strong> laminin is necessary for the MNPs toadhere and survive––MotorBlast and NeuroBlast both have a one monthshelf life84North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Pluripotent Stem CellsContinuedOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeFP-6011 FP-6011 Standard MotorPlate Human ESC Motor Neuron Progenitors 96-well plateFP-6046 FP-6046 Mature MotorPlate Human ESC Motor Neuron Progenitors 96-well plateFP-6096 FP-6096 Standard MotorPlate Human ESC Motor Neuron Progenitors 16 standard sample kit KitFP-6097 FP-6097 Mature MotorPlate Human ESC Motor Neuron Progenitors 16 mature sample kit KitFP-6098 FP-6098 Standard MotorPlate Human ESC Motor Neuron Progenitors 32 standard sample kit KitFP-6099 FP-6099 Mature MotorPlate Human ESC Motor Neuron Progenitors 32 mature sample kit KitFP-6051 FP-6051 Human ESC-derived Motor Neuron Progenitors Cryopreserved ≥4 million cells/vial100 ml bottle media included <strong>with</strong> each plate purchase.Ordering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeFP-6025 FP-6025 MotorBlast Motor Neuron Progenitors Culture Medium 100 mlFP-6059 FP-6059 MotorBlast Motor Neuron Progenitors Culture Medium 500 mlFP-6072 FP-6072 NeuroBlast Motor Neuron Progenitors Plating Medium For plating Human ESC-derived Motor Neuron Progenitors (FP-6051) 50 ml1Primary Cells and Media / Poietics Stem Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com85
Human Preadipocyte Cells and Media1Primary Cells and Media / Poietics Stem Cells and MediaPoietics Preadipocyte Cells are isolated from subcutaneousor visceral fat. Subcutaneous fat is often found attached toskin in the lower abdomen area. Visceral preadipocytes areisolated from adipose tissue associated <strong>with</strong> internalorgans, such as the bladder or kidney.Relative to subcutaneous fat, visceral fat deposits aremobilized at a higher rate to produce serum fatty acidswhich contribute to insulin resistance, Diabetes Type 2, andother related cardiovascular disorders.Preadipocytes are precursor cells that develop intoadipocytes when fully differentiated. Adipocytes performessential functions of energy metabolism and arecharacterized by the accumulation of intracellulartriglycerides.Poietics Preadipocyte Cells and Media System isComprised of:––Cryopreserved human preadipocyte cells isolated fromsubcutaneous or visceral fat––Cells available from normal, or Type I and Type II diabeticdonors––PGM-2 Preadipocyte Growth Medium-2 BulletKit,which contains the basal medium and growthsupplements needed to induce growth anddifferentiation of the preadipocytes into matureadipocytes––AdipoRed Assay Reagent, an assay reagent for highthroughputquantification of intracellular lipid■■Applications––Lipid accumulation andmetabolism––Obesity––Diet drug development––Diabetes research––Insulin sensitivityDifferentiated subcutaneous Differentiated visceral preadipocytepreadipocyte cellscells stained <strong>with</strong> Oil Red OCatecholamine-induced Lipolysis in Subcutaneous andt l i L S b ta uVisceral Prima Prima y uman Pread y Human pocytes Preadipocytes2.521.510.50SubcutaneousVisceralTotal stored triglycerideLipolysis (Glycerol)Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizePreadipocytesPT-5001 PT-5001 Human Subcutaneous Preadipocyte Cells Cryopreserved ≥4 million cells/vialPT-5005 PT-5005 Human Visceral Preadipocyte Cells Cryopreserved ≥1 million cells/vialPT-5020 PT-5020 Human Subcutaneous Preadipocyte Cells Cryopreserved ≥1 million cells/vialProgenitor and Stem Cells – DiseasedPT-5021 PT-5021 Diseased Human Subcutaneous Preadipocyte Cells, Diabetes Type I Cryopreserved ≥1 million cells/vialPT-5022 PT-5022 Diseased Human Subcutaneous Preadipocyte Cells, Diabetes Type II Cryopreserved ≥1 million cells/vialPT-5023 PT-5023 Diseased Human Visceral Preadipocyte Cells, Diabetes Type I Cryopreserved ≥1 million cells/vialPT-5024 PT-5024 Diseased Human Visceral Preadipocyte Cells, Diabetes Type II Cryopreserved ≥1 million cells/vial86North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Preadipocyte Cells and MediaContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-8002 PT-8002 PGM-2 Preadipocyte Growth Medium-2 BulletKit Includes basal medium and SingleQuots Kit KitPT-8202 PT-8202 PBM-2 Preadipocyte Basal Medium-2 500 mlPT-9502 PT-9502 PGM-2 Preadipocyte Growth Medium SingleQuotsKitSupplements and Growth FactorsPT-7009 PT-7009 AdipoRed Assay Reagent 5 × 4 ml17-512F BE17-512F Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO4) <strong>with</strong>out calcium or magnesium 500 mlSee pages 411–433.Related ProductsHuman Adipose-derived Stem Cells 77AdipoLyze Lipolysis Detection Assay 260Page1Primary Cells and Media / Poietics Stem Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com87
Human Mesenchymal Stem Cells and Media (hMSC)1Primary Cells and Media / Poietics Stem Cells and MediaBone marrow contains a population of rare progenitor cellsknown as mesenchymal stem cells (MSC) capable ofreplication as undifferentiated cells or differentiating intobone, cartilage, fat, muscle, tendon and marrow stroma.Poietics Human Mesenchymal Stem Cell System containsnormal human mesenchymal stem cells and medium fortheir growth. Cells are frozen in passage two and itsrecommended that experiments are performed by passagefive.Each system can generate hMSC cultures for experimentalstudies in cell differentiation, including osteogenesis andbone mineralization, chondrogenesis and cartilageformation, adipogenesis and fat accumulation. They areexcellent models for gene delivery research, functionalgenomics, drug screening, high-throughput screening andtoxicology.Poietics Cells and Media are quality tested and guaranteedto give optimum performance. hMSCs are tested using invitro assays and are found to be positive for: adipogeniclineage as indicated by neutral red stain; chondrogeniclineage as indicated by collagen type II and osteogeniclineage as indicated by calcium deposition.■■Cell Testing and Specifications––HIV-1, Hepatitis B and Hepatitis C are not detected for alldonors––Cells are tested for purity by flow cytometry:––Cells are positive for CD105, CD166, CD29, and CD44––Cells are negative for CD14, CD34, and CD45––Tested for the ability to differentiate into osteogenic,chondrogenic, adipogenic lineageshMSC in cultureStromal cell culture1,0008006004002000200 400 600 800 1,000Side Scatter1,000Poietics Human MSCMesenchymal stem cells are licensed by Lonza from Osiris Therapeutics,Inc. and are subject to the following limited use license:The included biological material, including progeny and derivatives,(collectively referred to as Material) is licensed to you under specific terms.You are responsible for ensuring that the terms of the license agreementare met.1. Grants of License: Lonza Walkersville, Inc. grants you anon-transferable, non-exclusive license to use the Material forresearch.2. Not for Human Use: The Material may not be used: a) in humans; b) inconjunction <strong>with</strong> human clinical trials; c) in association <strong>with</strong> humandiagnostics.3. Material Not Transferable: You may not transfer the Material to anyother person or organization.4. Patent Notice: Material under license from Osiris Therapeutics, Inc.Material is covered by US patent 5,486,359 and others.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size8006004002000200 400 600 800 1,000Side ScatterPT-2501 PT-2501 hMSC – Human Mesenchymal Stem Cells Cryopreserved ≥750,000 cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-3001 PT-3001 MSCGM Mesenchymal Stem Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit KitPT-3002 PT-3002 hMSC – Human Mesenchymal Stem Cell OsteogenicIncludes basal medium and SingleQuots Kit KitDifferentiation Medium BulletKitPT-3003 PT-3003 hMSC – Human Mesenchymal Stem Cell ChondrogenicDifferentiation Medium BulletKitIncludes basal medium and SingleQuots Kit,TGF- 3 sold separatelyKit88North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Mesenchymal Stem Cells and Media (hMSC)ContinuedOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizePT-3004 PT-3004 hMSC – Human Mesenchymal Stem Cell AdipogenicIncludes basal medium and SingleQuots Kit KitDifferentiation Medium BulletKitPT-3238 PT-3238 MSCBM Mesenchymal Stem Cell Basal Medium 440 mlPT-4105 PT-4105 MSCGM Mesenchymal Stem Cell Growth MediumKitSingleQuots Supplements and Growth FactorsPT-4124 PT-4124 rhTGF-ß3 for hMSC Chondrogenic Differentiation MediumSupplement2 µgOrdering Information – ReagentsCat. No. NA Cat. No. EU Product Name Product Description Size17-512F BE17-512F Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO4) <strong>with</strong>out calcium or magnesium 500 mlSizeCC-3232 CC-3232 Trypsin/EDTA for Mesenchymal Stem Cells 100 mlRelated ProductsRat Mesenchymal Stem Cells 90Rat Mesenchymal Stem Cell Growth Medium BulletKit 90OsteoImage Mineralization Assay 263AdipoRed Assay Reagent 259Adipolyze Lipolysis Detection Assay 260Nucleofector Kits for Primary Stem Cells 222Page1Primary Cells and Media / Poietics Stem Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com89
Pluripotent Stem Cell Services1Lonza established a new strategic vision to become the leading supplier to the regenerative medicine industry. To realize thisvision, Lonza created the Pluripotent Stem Cell Innovation Center. PSCs have the ability to generate any of the220+ cell types in the human body. Consequently, these cells have great potential in basic research, drug discovery andregenerative medicine.Primary Cells and Media / Poietics Stem Cells and MediaiPSCsTissue AcquistionReprogrammingLonza has built up expertise, capacity, and capabilities in pluripotent stem cell research and their application to cGMP manufacturing. <strong>Research</strong>ers can now accessthis expertise through our PSC service offering from iPSC generation to process development and differentiation.Our services span the full value chain of pluripotent stemcells from tissue acquisition to differentiation:■■Tissue Acquisition – We have a dedicated team thatprocures both research and cGMP grade tissueaccording to the highest ethical standards and incompliance <strong>with</strong> government regulations.■■Reprogramming – We offer cGMP and non-cGMP iPSCgeneration under feeder- and feeder-free conditionsusing a zero-footprint technology■■Growth / Expansion / Banking – We have establishedprotocols using all of the common medium, matrix, andpassaging methods. We also have the infrastructureand resources to support small- and large-scale cultureand banking of PSCs.■■Characterization – We offer all the standard methods ofcharacterizing PSCs including thawing efficiency,myoplasma and sterility testing, karyotype analysis,short tandem repeat genotyping, pluripotency markerexpression (flow cytometry and immunofluorescence),and pluripotency assays (embryoid body and teratomaformation).■■Differentiation – We have established protocols for theproduction of PSC-derived motor neurons, dopaminergicneurons, and neural stem cells. We also havedevelopment programs underway to add to ourdifferentiation portfolio of therapeutically relevant celltypes■■Process development – Over the years we have built upexpertise in the differentiation of high purity, functionalcell types. Our team is well versed in technology transferand optimization of manufacturing protocolsGrowth ExpansionGrowth ExpansionAlkalinePhosphateAlkalinePhosphateCharacterizationlCharacterizationlOCT4 SSEA4NANOG DAPIDifferentiationDifferentiationHuman induced pluripotent stem cells express hESC-associated markersPOU5F1/OCT4 (green) and SSEA4 (red) counterstained <strong>with</strong> DAPI (blue).90North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Rat Mesenchymal Stem Cells and Media (rMSC)Poietics Rat Mesenchymal Stem Cells are isolated frombone marrow taken from the femur and tibia of femaleFischer 344 rats. These rMSCs test positive for surfacemarkers CD29 and CD90, and negative for CD11b, CD34,and CD45 by flow cytometry. Cells also test positive usingin vitro assays for adipogenesis indicated by Oil Red O, andosteogenesis indicated by calcium mineralization.Optimized media kits are available for growth and expansionof the cells as well as osteogenic and adipogenicdifferentiation. Poietics Cells and Medium are qualitytested and guaranteed to give optimal performance.■■Benefits––Performance tested as a complete system––Guaranteed to proliferate through several morepassages––Cells retain their plasticity in culture––Cells can differentiate into bone, cartilage, fat, muscle,tendon and marrow stroma■■Applications––Cell differentiation––Drug screening and toxicology––Functional genomics––Development of cell-based disease therapies■■Cell Testing and Specifications––Isolated from the bone marrow of Fischer 344 rats––Cryopreserved at P3––Positive for CD29 and CD90 surface markers––Adipogenic and osteogenic differentiation––Negative for mycoplasma, bacteria, and fungiRat osteoblasts after 4 weeks in culture <strong>with</strong> Rat MSC OsteogenicDifferentiation BulletKit. Rat MSCs stained <strong>with</strong> OsteoImage Assay forbone mineralization (osteogenesis).Rat MSCs differentiated into adipocytes stained <strong>with</strong> Oil Red O.1Primary Cells and Media / Poietics Stem Cells and MediaOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizePT-2505 PT-2505 R-MSC – Rat Mesenchymal Stem Cells Cryopreserved ≥750,000 cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description Size00192853 00192853 Rat MSCGM Mesenchymal Stem Cell Growth MediumBulletKit00192854 00192854 rMSC – Rat Mesenchymal Stem Cell OsteogenicDifferentiation BulletKit00192855 00192855 rMSC – Rat Mesenchymal Stem Cell AdipogenicDifferentiation BulletKitIncludes basal medium and SingleQuots Kit500 ml170 ml170 mlRelated ProductsNucleofector Kits for Primary Stem Cells 222PageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com91
Poietics Immune Cells and MediaLeading the attack on immune cell research1Primary Cells and Media / Poietics Stem Cells and MediaPoietics Immune Cells and MediaIntroduction93Human Dendritic Cells and Media 94Human CD14 + Monocytes and Media 95Human Natural Killer Cells96Human T Cells and Media 9692North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionPoietics Primary Human Immune Cells and Media areisolated from either peripheral blood or cord blood fromhealthy donors. Each donor is tested and found non-reactiveby an FDA approved method for the presence of HIV-I,Hepatitis B Virus and Hepatitis C Virus. Where donor testingis not possible, cell products are tested for the presence ofviral nucleic acid from HIV, Hepatitis B Virus, and Hepatitis CVirus. Poietics Primary Human Immune Cells purity levelsare verified via flow cytometric analysis.Poietics Primary Human Immune Cells and Media can beused in various immunological research applications,including:––Transplantation––Autoimmune diseases––HIV/AIDS––Cellular immunity––Immunotherapy––Cancer––Cell memory––Histocompatability––Graft-versus-host disease1Primary Cells and Media / Poietics Immune Cells and MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com93
Human Dendritic Cells and Media (NHDC)1Primary Cells and Media / Poietics Immune Cells and MediaPoietics Normal Human Dendritic Cells and MediaConsist of:––Cryopreserved primary derived cultures of – normalhuman dendritic cells derived from human peripheralblood mononuclear cells––LGM-3 Lymphocyte Growth Medium-3, a serum-freemedium for lymphocyte cell growthAll cells are isolated from peripheral blood by apheresis anddensity gradient centrifugation. Dendritic cells, andperipheral blood mononuclear cells, are available from thesame donor.Poietics Normal Human Dendritic Cells do not proliferateand can survive up to 7 days in culture <strong>with</strong> the propercytokines (IL-4 and GM-CSF). Poietics Human PeripheralBlood Mononuclear Cells cells, when properly stimulated,can divide in culture.■■Applications––Antigen presentation and uptake––Pharmacology––Drug-induced immunosuppression of cytokinesecretionNormal human dendritic cellsOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description SizeCC-2701 CC-2701 NHDC – Human Dendritic Cells Cryopreserved, in LGM-3 BulletKit ≥2.5 million cells/vialCC-2702 CC-2702 HPBMC – Human Peripheral Blood Mononuclear Cells Cryopreserved, volume discount available ≥50 million cells/vialPlease inquire about availability of NHDC and HPBMC matched sets from the same donorOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3211 CC-3211 LGM-3 Lymphocyte Growth Medium 500 mlRelated ProductsNucleofector Kits for Primary Blood Cells 187Page94North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human CD14 + Monocytes and MediaNormal human monocytes are found in the circulatingperipheral blood. They play an important role in hostdefense as circulating monocytes and differentiation intotissue macrophages, and can differentiate into dendriticcells <strong>with</strong> potent antigen-presenting capability, in vivo andin vitro.Monocytes are isolated from the peripheral blood ofscreened, healthy donors. Peripheral blood is collected fromthe donors using apheresis. The cell product, enriched formononuclear cells, is further processed by densitycentrifugation to remove the remaining red cells andneutrophils. Monocytes are then isolated using positiveimmunomagnetic selection directed against CD14.Media RecommendationsTo maintain the monocyte phenotype, serum-containingmedium is recommended (10% FBS is generallyrecommended). M-CSF (at 10 ng/ml) can also be added. Toproduce osteoblasts, both soluble RANK ligand (30 ng/ml)and M-CSF (30 ng/ml) need to be added to serumcontainingmedium. To produce dendritic cells, a serum-freemedium, such as LGM-3 Lymphocyte Growth Medium-3should be used, <strong>with</strong> the addition of GM-CSF(50 ng/ml) and IL-4 (50 ng/ml).Human CD14 + monocytesOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size2W-400C 2W-400C Human Peripheral Blood CD14 + Monocytes Cryopreserved ≥10 million cells/vial2W-400B 2W-400B Human Peripheral Blood CD14 + Monocytes Cryopreserved ≥20 million cells/vial2W-400A 2W-400A Human Peripheral Blood CD14 + Monocytes Cryopreserved ≥40 million cells/vial1Primary Cells and Media / Poietics Immune Cells and MediaOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3211 CC-3211 LGM-3 Lymphocyte Growth Medium 500 mlRelated ProductsNucleofector Kits for Primary Blood Cells 187PageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com95
Human Natural Killer (NK) Cells1Primary Cells and Media / Poietics Immune Cells and MediaHuman natural killer cells are lymphocytes of the immunesystem that are critical in host defense and immuneregulation. Since they are part of innate immunity, they donot require sensitization for the expression of their activity.NK cells play significant roles in viral infections,autoimmunity, pregnancy, cancer, bone marrowtransplantation, and more recently, adaptive immunity. NKcells are most traditionally characterized by the presenceof surface marker CD56, and absence of CD3. PoieticsHuman Natural Killer Cells are isolated using positive ornegative immunomagnetic selection. Purity is ≥90% viaflow cytometry based on the presence of the CD56 antigen.CD16 expression is also reported. Negative selection of NKcells results in significantly greater enrichment of CD16+cells (usually >80% of total cells), than does positiveHuman T Cells and MediaPoietics Human CD4+ T Cells are a type of lymphocyte thatplay an important role in the immune system since theylead attack against infections. These cells are also referredto as helper cells. These helper cells orchestrate the body’sresponse to certain microorganisms, such as viruses.Poietics CD4+ T Cells are isolated from normal peripheralblood mononuclear cells using negative immunomagneticselection of the CD4 antigen. Purity is ≥ 90%.Human natural killer cellselection which has much greater variability for CD16+cells. Cells isolated via positive selection are also referred toas natural killer T cells.Ordering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size2W-501 2W-501 NK – Human Natural Killer Cells Cryopreserved, negative selection ≥5 million cells/vial2W-502 2W-502 NK – Human Natural Killer Cells Cryopreserved, positive selection ≥5 million cells/vial■■Applications––Immunotherapy––Inflammatory response––AutoimmunityOrdering Information – CellsCat. No. NA Cat. No. EU Product Name Product Description Size2W-200 2W-200 Human Peripheral Blood CD4 + T Cells Cryopreserved ≥10 million cells/vialOrdering Information – MediaCat. No. NA Cat. No. EU Product Name Product Description SizeCC-3211 CC-3211 LGM-3 Lymphocyte Growth Medium 500 mlRelated ProductsNucleofector Kits for Primary Blood Cells 187–197Page96North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>2 Media, Reagents and Sera2BioWhittaker Classical Media 99BioWhittaker Specialty Media 113BioWhittaker Cell Culture Reagents 135BioWhittaker Animal and Human Sera 145
Media, Reagents and Sera2Media, Reagents and Sera / BioWhittaker Classical MediaBioWhittaker Classical MediaIntroduction100Basal Medium Eagle 102Dulbecco’s Modified Eagle’s Medium 102DMEM:F12 Medium 104Glasgow Minimum Essential Medium 104Grace’s Insect Medium 105Ham’s Medium 105Iscove’s Modified Dulbecco’s Medium 106L-15 Medium106McCoy’s 5A Medium 107Minimum Essential Medium – Eagle 107Medium 199 109NCTC-109 Medium 109Richter’s CM 109RPMI 1640 Medium 110Insect Medium 111Waymouth’s Medium 111William’s Medium 111BioWhittaker Specialty MediaIntroduction114TheraPEAK MSCGM-CD Mesenchymal Stem CellGrowth Medium – Chemically-Defined 115FGM-CD Fibroblast Growth Medium –Chemically Defined 116UltraCULTURE Serum-free Medium 117PC-1 Chemically Defined, Serum-free Medium –Chemically Defined 118UltraMEM Reduced Serum Media 119X-VIVO Serum-free Hematopoietic Cell Media –Chemically Defined 120UltraCHO Serum-free CHO Cell Media 121ProCHO Protein-free CHO Media 122PowerCHO Serum-free CHO Media –Chemically Defined 123ProFreeze-CDM, NAO Freeze Medium –Chemically Defined 124UltraMDCK Serum-free Renal Cell Medium –Chemically Defined 124Pro293 Serum-free Media – Chemically Defined 125ProVero 1 Serum-free Medium 125ProPer 1 Serum-free Medium – Chemically Defined 126PERMEXCIS® Virus Production Medium –Chemically Defined 127Lymphochrome Medium 127Amniochrome II Modified Medium 128Amniochrome Plus Medium 128Amniochrome Pro Medium 128HL-1 Serum-free Media – Chemically Defined 129UltraDOMA Serum-free Hybridoma Medium 131ProDoma Serum-free Hybridoma Media –Chemically Defined 132UltraDOMA-PF Protein-free Hybridoma Media 132Insect-XPRESS Protein-free Insect Cell Medium 133ProNS0 Protein-free Media – Chemically Defined 134BioWhittaker Cell Culture ReagentsIntroduction136Balanced Salt Solutions 137Earle’s BSS 137Hanks’ BSS 137Reagents138Growth Factors 140Antibiotics and Antimycotics 140Penicillin-Streptomycin Mixtures 141Buffers and Buffered Salines 142Viral Serology 144BioWhittaker Animal and Human SeraIntroduction146Fetal Bovine Sera 149Bovine Sera 149Human Sera 150Fetal Bovine Serum 150Equine Sera 151For further manufacturing or laboratory use. Not for use in diagnostic procedures.98North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
BioWhittaker Classical Media2BioWhittaker Classical MediaIntroduction100Basal Medium Eagle 102Dulbecco’s Modified Eagle’s Medium 102DMEM:F12 Medium 104Glasgow Minimum Essential Medium 104Grace’s Insect Medium 105Ham’s Medium 105Iscove’s Modified Dulbecco’s Medium 106L-15 Medium106McCoy’s 5A Medium 107Minimum Essential Medium – Eagle 107Medium 199 109NCTC-109 Medium 109Richter’s CM 109RPMI 1640 Medium 110Insect Medium 111Waymouth’s Medium 111William’s Medium 111Media, Reagents and Sera / BioWhittaker Classical MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com99
Introduction2Liquid Cell Culture MediaChemically defined liquid media are used to providenutrients for cell culture growth in research, diagnostic, andmanufacturing applications. In order to meet your specificneeds, our production and quality control procedures forliquid media emphasize control. Our goal is to provide you<strong>with</strong> high quality products that are consistent from batch tobatch. With all of the time that you take to optimize yoursystems, we want to provide you <strong>with</strong> products you cantrust.All BioWhittaker Classical Media products are manufacturedin accordance <strong>with</strong> cGMP regulations. A Device MasterRecord (DMR) is prepared for every liquid medium product.It defines the procedures for production from receipt of rawmaterials to final product release, the environmental andprocessing controls required, as well as productspecifications, including packaging and labeling. Productmanufacturing is consistent <strong>with</strong> the requirements definedin the DMR, which ensures that each lot of a product isconsistent <strong>with</strong> all other lots of the same product.Chemicals used to prepare liquid media products arepurchased according to the raw material qualifications fromapproved suppliers. Each lot must meet establishedcomponent specifications before it is released by QualityAssurance for use. We manufacture all liquid cell culturemedia using Water for Injection (WFI) quality water, whichhas been prepared by ultrafiltration, reverse osmosis,deionization, and distillation. Liquid media is sterile filteredthrough pharmaceutical-grade sterilizing filters. The for -mulations used for standard liquid media products arethose recommended by the Tissue Culture Association.Quality ControlIn order to maintain consistent quality in sterile cell culturemedia products, strict quality control of each production lotis essential. Written procedures in accordance <strong>with</strong> currentGood Manufacturing Practices (cGMPs) provide qualitycontrol from start to finish for each product produced. Finalproduct testing includes the following:USP Sterility – Tests are performed on representativesamples using the membrane filtration procedure inaccordance <strong>with</strong> the US Pharmacopoeia or EPPharmacopoeia. Culture media used are fluid thioglycollatemedium (FTM) and trypticase soy broth (TSB). All testedmedia must pass growth promotion testing before use. Thetest samples are filtered and the filters are immersed inFTM and TSB. TSB cultures are incubated at 22.5°C ± 2.5°C.FTM cultures are incubated at 32.5°C ± 2.5°C. The culturesare incubated for 14 days, during which they are periodicallyexamined and sterility results are recorded.Chemistries (pH and osmolality) – Tests are performed onrepresentative samples from each lot using routinelycalibrated equipment. Osmolality is determined by meansof the highly repeatable freezing point method.Cell Growth Promotion – Media and supplements are testedfor their growth promoting properties using primary, diploid,and heteroploid cell lines unless other cells are appropriatebased on the product use. Results are obtained using theLowry protein method or manual cell count to assess cellreplication. Visual screening of cultures is also done toensure characteristic cell morphology and lack of toxicity.Additional testing for toxicity, function and/or activity mayalso be conducted if one or more is required in the productrelease specifications.100North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionContinuedEndotoxin – Products are tested for endotoxin contentusing the Kinetic-QCL Assay. Test dilutions used arescreened for inhibition and enhancement in the Kinetic-QCLAssay. Endotoxin levels for these products are available onCertificates of Analysis.The combined result of all in-process monitoring and finalproduct testing is the further assurance that each lot hasbeen prepared not only according to approved writtenprocedures, but has passed test criteria, and will meetdesign specifications. Samples from each lot of standardproduct are retained and stored at label temperature in theevent follow up testing is required. Subsequent testing maybe in response to your inquiries or for shelf life studies.Micronized Media (Powder)Some tissue culturists prefer to prepare their own liquidmedium from powder. Therefore, we are committed tooffering you the same high quality powdered media as youhave come to expect <strong>with</strong> our liquid media. We utilizeunique manufacturing processes, which produce thepowder in a controlled environment, guaranteeingmaximum stability and minimum degradation due tochemical oxidation. Standard package sizes are 10 l and50 l. Other sizes are available by contacting your local salesrepresentative.Quality Control TestingAll powdered media are subject to rigorous quality controltesting. These tests include: pH, osmolality, cell growth,endotoxin, moisture content, and a sieve test.Shelf LifePowdered media are generally very stable at refrigeratedtemperatures. Shelf life is two years on most powderedproducts.For guidelines on preparing powdered media, seepage 437.2Media, Reagents and Sera / BioWhittaker Classical MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com101
Basal Medium Eagle (BME)A minimal medium suitable for a variety of cell types. It is ahistorical precursor to Minimum Essential Medium (MEM).15°C–30°C2Ordering Information – Basel Medium EagleCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-132A 12-132A Cryoprotective Freezing Medium Without L-glutamine, <strong>with</strong> 15% DMSO (Use 1:1 <strong>with</strong> growth medium) 15°C–30°C 100 ml12-105F BE12-105F Basal Medium Eagle (BME) With Earle‘s BSS, <strong>with</strong>out L-glutamine 15°C–30°C 500 mlCryoprotective Medium – see ProFreeze-CDM, non-animal origin, chemically-defined freeze medium, Cat. No. 12-769E, page 124.For a basic procedure for cryopreservation of cells, see page 433Media, Reagents and Sera / BioWhittaker Classical MediaDulbecco’s Modified Eagle’s MediumDMEM is used in a wide range of mammalian cell cultureapplications. The high glucose version is well suited to highdensity suspension culture. The low glucose formula isused for adherent dependent cells.Cat. No.SizeWithoutL-glutamineWithSodiumPyruvateWithoutPhenol RedWithHEPES2°C–8°CWith1.0 g/LGlucoseWith4.5 g/LGlucose12-614F 500 ml ■ ■ ■12-614Q 1 l ■ ■ ■12-604F 500 ml ■ ■12-604Q 1 l ■ ■BE15-604F 50 l ■ ■BE15-604D 1 × 10 l ■ ■BE12-604F/U1 500 ml ■ ■ ■ ■12-914F 500 ml ■ ■ ■ ■12-917F 500 ml ■ ■ ■ ■12-707F 500 ml ■ ■ ■12-708F 500 ml ■ ■ ■ ■12-709F 500 ml ■ ■ ■ ■12-733F 500 ml ■ ■12-733Q 1 l ■ ■12-741F 500 ml ■PowderHybridomaScreenedWith UG I** With UltraGlutamine IFor exact compositions, see reagents formulation section, page 439–454.102North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Dulbecco’s Modified Eagle’s MediumContinuedOrdering Information – Dulbecco’s Modified Eagle MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-614F BE12-614F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong>out L-glutamine 2°C–8°C 500 mlBE12-604F/U1 BE12-604F/U1 Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong> UltraGlutamine 2°C–8°C500 mlI12-604F BE12-604F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong> L-glutamine 2°C–8°C 500 ml2Ordering Information – Dulbecco’s Modified Eagle MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-914F BE12-914F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong>out L-glutamine, 2°C–8°C500 mlhybridoma tested12-707F BE12-707F Dulbecco‘s Modified Eagle Medium (DMEM) With 1.0 g/l glucose, <strong>with</strong>out L-glutamine 2°C–8°C 500 ml12-733F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong>out L-glutamine 2°C–8°C500 mlor sodium pyruvate12-708F BE12-708F Dulbecco‘s Modified Eagle Medium (DMEM) With 1.0 g/l glucose, <strong>with</strong>out L-glutamine, 2°C–8°C500 ml<strong>with</strong> 25 mM HEPES12-741F BE12-741F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong> L-glutamine, 2°C–8°C500 ml<strong>with</strong>out sodium pyruvate12-917F Dulbecco‘s Modified Eagle Medium (DMEM) With 4.5 g/l glucose, <strong>with</strong>out L-glutamine 2°C–8°C500 mlor phenol redBE15-604D BE15-604D Dulbecco‘s Modified Eagle Medium (DMEM), Powder With 4.5 g/l glucose <strong>with</strong> L-glutamine, 2°C–8°C10 l<strong>with</strong>out sodium pyruvateBE15-604F BE15-604F Dulbecco‘s Modified Eagle Medium (DMEM), Powder With 4.5 g/l glucose <strong>with</strong> L-glutamine,<strong>with</strong>out sodium pyruvate2°C–8°C50 lMedia, Reagents and Sera / BioWhittaker Classical MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com103
DMEM:F12 MediumDMEM:F12 combines the richness of F12 <strong>with</strong> the highercomponent concentrations of DMEM. This medium is well –suited for clonal density cultures.WithoutL-glutamineWithHEPESWith3.151 g/LGlucoseWith UG I*Powder2Media, Reagents and Sera / BioWhittaker Classical Media2°C–8°CGlasgow Minimum Essential Medium (GMEM)Designed to support BHK-21 cells.Cat. no.Size15-719D 1 × 10 l ■ ■ ■12-719F 500 ml ■ ■12-719Q 1 l ■ ■BE04-687Q 1 l ■BE04-687F/U1 500 ml ■ ■ ■2°C–8°CFor exact compositions, see reagentsformulation section, page 439–455.Ordering Information – Dulbecco’s Modified Eagle Medium:F12 (DMEM:F12)Cat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-719F BE12-719F Dulbecco‘s Modified Eagle Medium:F12 (DMEM:F12) 1:1 mixture <strong>with</strong> 3.151 g/l glucose,L-glutamine and 115 mM HEPES12-719Q 12-719Q Dulbecco‘s Modified Eagle Medium:F12 (DMEM:F12) 1:1 mixture <strong>with</strong> 3.151 g/l glucose,L-glutamine and 115 mM HEPESBE04-687F/U1 BE04-687F/U1 Dulbecco‘s Modified Eagle Medium:F12 (DMEM:F12) 1:1 mixture <strong>with</strong> 3.151 g/l glucose, <strong>with</strong>UltraGlutamine I, <strong>with</strong>out HEPESBE04-687Q BE04-687Q Dulbecco‘s Modified Eagle Medium:F12 (DMEM:F12) 1:1 mixture <strong>with</strong> 3.151 g/l glucose, <strong>with</strong>UltraGlutamine I, <strong>with</strong>out HEPES2°C–8°C2°C–8°C2°C–8°C2°C–8°COrdering Information – Glasgow Minimum Essential MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-739F BE12-739F Glasgow Minimum Essential Medium With L-glutamine 2°C–8°C 500 ml500 ml1 l500 ml1 l104North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Grace’s Insect MediumClassical insect cell media.2°C–8°COrdering Information – Grace’s Insect MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-457F 04-457F Grace‘s Insect Medium Without FBS, <strong>with</strong> yeastolate, lactalbumin hydrolysate orgentamicin04-501Q 04-501Q Grace‘s Complete Insect Medium (TNM-FH) With 10% heat-inactivated FBS, yeastolate, lactalbuminhydrolysate, and gentamicin04-649F 04-649F Grace‘s Insect Medium Without FBS, <strong>with</strong> yeastolate, lactalbumin hydrolysate,and gentamicin2°C–8°C2°C–8°C2°C–8°C500 ml1 l500 ml2Related ProductsInsect-XPRESS Protein-free Insect Cell Medium 133Schneider‘s Drosophila Medium 111ITC 100 Insect Medium 111Ham’s MediumDeveloped for low density (clonal) growth of CHO cells.2°C–8°COrdering Information – Ham’s F10 MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-014F Ham‘s F10 Medium With UltraGlutamine I, <strong>with</strong>out thymidine 2°C–8°C 500 ml12-615F BE12-615F Ham‘s F12 Medium With L-glutamine 2°C–8°C 500 ml12-618F BE12-618F Ham‘s F10 Medium With L-glutamine 2°C–8°C 500 ml12-615Q 12-615Q Ham‘s F12 Medium With L-glutamine 2°C–8°C 1 lPageMedia, Reagents and Sera / BioWhittaker Classical MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com105
Iscove’s Modified Dulbecco’s Medium (IMDM)IMDM is suitable for fast growing cells. All formulas containHEPES for added buffering.WithoutL-glutamineWithHEPESHybridomaScreenedPowder2°C–8°CCat. No.Size212-722F 500 ml ■12-722Q 1 l ■12-726F 500 ml ■ ■12-726Q 1 l ■ ■12-915F 500 ml ■ ■Media, Reagents and Sera / BioWhittaker Classical MediaOrdering Information – Iscove’s Modified Dulbecco’s Medium (IMDM)Cat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-915F BE12-915F Iscove‘s Modified Dulbecco‘s Medium (IMDM) With L-glutamine and 25 mM HEPES, hybridoma tested 2°C–8°C 500 ml12-722F BE12-722F Iscove‘s Modified Dulbecco‘s Medium (IMDM) With L-glutamine and 25 mM HEPES 2°C–8°C 500 ml12-722Q 12-722Q Iscove‘s Modified Dulbecco‘s Medium (IMDM) With L-glutamine and 25 mM HEPES 2°C–8°C 1 l12-726F BE12-726F Iscove‘s Modified Dulbecco‘s Medium (IMDM) Without L-glutamine, <strong>with</strong> 25 mM HEPES 2°C–8°C 500 ml12-726Q 12-726Q Iscove‘s Modified Dulbecco‘s Medium (IMDM) Without L-glutamine, <strong>with</strong> 25 mM HEPES 2°C–8°C 1 lL-15 (Leibovitz) MediumDeveloped for fast growing tumor cells, this formula doesnot require a CO 2enriched atmosphere. The bicarbonatefreemedium is buffered <strong>with</strong> elevated levels of amino acids.2°C–8°COrdering Information – L-15 Leibovitz’s MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-700Q 12-700Q L-15 Leibovitz‘s Medium Without L-glutamine 2°C–8°C 1 l12-700F 12-700F L-15 Leibovitz‘s Medium Without L-glutamine 2°C–8°C 500 ml12-669E 12-669E L-15 Leibovitz‘s Modified Medium (2X) (2X) except L-tyrosine (1X), <strong>with</strong>out L-glutamine orphenol red (virus plaquing medium)2°C–8°C100 ml106North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
McCoy’s 5A MediumDesigned for human lymphocyte culture.2°C–8°COrdering Information – McCoy’s 5A MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-168F BE12-168F McCoy‘s 5A Medium With L-glutamine and 25 mM HEPES 2°C–8°C 500 ml12-688F BE12-688F McCoy‘s 5A Medium With L-glutamine 2°C–8°C 500 ml2Minimum Essential Medium – Eagle (MEM Eagle or E-MEM)MEM Eagle is suitable for a diverse spectrum of mammaliancell types. It is available <strong>with</strong> either Hanks’ or Earle’s salts.MEM-Hanks’ (12-127F or 12-137F) does not require a CO 2enriched atmosphere. Joklik’s modification is intended forsuspension culture.Cat. No.SizeWithL-glutamineWithSodiumPyruvateWithoutPhenol RedWithHEPESWithHBSSWithEBSS12-169F (Alpha) 500 ml ■ ■ ■2°C–8°C unless noted otherwise in the orderinginformationPowderWith NEAAWithPen-strepamphBBE02-002F 500 ml ■ ■ ■ ■ ■15-611D 1 × 10 l ■ ■ ■ ■12-611F 500 ml ■ ■12-611Q 1 l ■ ■12-125F 500 ml ■12-125Q 1 l ■12-662F 500 ml ■ ■ ■12-662Q 1 l ■ ■ ■12-136F 500 ml ■ ■12-136Q 1 l ■ ■12-736E 100 ml ■ ■ ■ ■ ■12-736F 500 ml ■ ■ ■ ■ ■12-684F (10x) 500 ml ■12-668E (2x) 100 ml ■ ■06-174G 450 ml ■ ■ ■ ■12-127F 500 ml ■12-137F 500 ml ■ ■04-719Q (Joklik’s) 1 l ■ ■ ■Without NabicarbonateWithoutCalciumWith UG I*Media, Reagents and Sera / BioWhittaker Classical MediaOrdering information on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com107
Minimum Essential Medium – Eagle (MEM Eagle or E-MEM)Continued2°C–8°C unless noted otherwise in the orderinginformation2Media, Reagents and Sera / BioWhittaker Classical MediaOrdering Information – Minimum Essential Medium – Alpha – EagleCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-002F BE02-002F Minimum Essential Medium – Alpha – Eagle With UltraGlutamine I, deoxyribonucleoside andribonucleosides12-169F BE12-169F Minimum Essential Medium – Alpha Eagle<strong>with</strong> Earle‘s BSSWithout L-glutamine, deoxyribonucleosides andribonucleosides2°C–8°C2°C–8°C12-662F BE12-662F Minimum Essential Medium – Eagle Without L-glutamine, <strong>with</strong> non-essential amino acids 2°C–8°C500 mland sodium pyruvate12-662Q 12-662Q Minimum Essential Medium – Eagle Without L-glutamine, <strong>with</strong> non-essential amino acids 2°C–8°C1 land sodium pyruvate12-137F 12-137F Minimum Essential Medium – Eagle Without L-glutamine, <strong>with</strong> 25 mM HEPES 15°C–30°C 500 mlBE15-611D BE15-611D Minimum Essential Medium – Eagle <strong>with</strong> With L-glutamine 2°C–8°C 10 lEarle‘s BSS12-611F BE12-611F Minimum Essential Medium – Eagle <strong>with</strong> With L-glutamine 2°C–8°C 500 mlEarle‘s BSS12-136Q BE12-136Q Minimum Essential Medium – Eagle <strong>with</strong> Without L-glutamine, <strong>with</strong> 25 mM HEPES 15°C–30°C 1 lEarle‘s BSS12-136F BE12-136F Minimum Essential Medium – Eagle <strong>with</strong> Without L-glutamine, <strong>with</strong> 25 mM HEPES 15°C–30°C 500 mlEarle‘s BSS12-125F BE12-125F Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSSWithout L-glutamine 15°C–30°C 500 ml06-174G BE06-174G Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS12-736F 12-736F Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS12-736E 12-736E Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS12-611Q 12-611Q Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS12-125Q 12-125Q Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS12-684F BE12-684F Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS (10X)BE12-668F BE12-668F Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS (2X)12-668E 12-668E Minimum Essential Medium – Eagle <strong>with</strong>Earle‘s BSS (2X)12-127F BE12-127F Minimum Essential Medium – Eagle <strong>with</strong>Hank‘s BSS04-719Q 04-719Q Minimum Essential Medium – Eagle Joklik’sFormulation500 ml500 mlWith non-essential amino acids, and L-glutamine, 2°C–8°C450 ml<strong>with</strong>out calciumWith L-glutamine 2°C–8°C 500 mlCell culture maintenance medium 12-136 <strong>with</strong> nonessentialamino acids, L-glutamine, 25 mM HEPES,10 μg/ml gentamicin, 50 units/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B, and 2.0%heat-inactivated FBS2°C–8°C100 mlWith L-glutamine 2°C–8°C 1 lWithout L-glutamine 15°C–30°C 1 lWithout L-glutamine or NaHCO3 15°C–30°C 500 mlWithout L-glutamine or phenol red (virus plaquingmedium)15°C–30°C500 mlWithout L-glutamine or phenol red (virus plaquing 15°C–30°C100 mlmedium)Without L-glutamine 15°C–30°C 500 mlFor suspension cultures, <strong>with</strong> L-glutamine, <strong>with</strong>outcalcium2°C–8°C1 l108North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Medium 199Medium 199 was originally formulated for chick embryofibroblast culture. These four formulations require a CO 2enriched atmosphere.WithL-glutamineWith HEPESWith HBSSWith EBSSWith 1.4 g/LNa-bicarbonateWith 2.2 g/LNa-bicarbonate2°C–8°CCat. No.SizeNCTC-109 MediumA complex formula used to supplement hybridoma medium.12-117F 500 ml ■ ■ ■ ■12-117Q 1 l ■ ■ ■ ■12-118F 500 ml ■ ■ ■ ■12-119F 500 ml ■ ■ ■12-109F 500 ml ■ ■ ■For exact compositions, see reagents formulation section, page 439–455Ordering Information – Medium 199Cat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-109F BE12-109F Medium 199 With Hank‘s BSS, L-glutamine, and 1.4 g/l NaHCO3 2°C–8°C 500 ml12-117F BE12-117F Medium 199 With Earle‘s BSS, L-glutamine, 25 mM HEPES, and 2.2 g/l NaHCO3 2°C–8°C 500 ml12-118F BE12-118F Medium 199 With L-glutamine, 25 mM HEPES, and 1.4 g/l NaHCO3 2°C–8°C 500 ml12-119F BE12-119F Medium 199 With Earle‘s BSS, L-glutamine, and 2.2 g/l NaHCO3 2°C–8°C 500 ml2°C–8°COrdering Information – NCTC-109 MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-923E 12-923E NCTC-109 Medium With Earle‘s BSS and L-glutamine, hybridoma screened 2°C–8°C 100 ml2Media, Reagents and Sera / BioWhittaker Classical MediaRichter’s CMA complex formula used to supplement hybridoma medium.2°C–8°COrdering Information – Richter’s CM MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-648F 04-648F Richter‘s CM Medium With L-glutamine 2°C–8°C 500 mlEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com109
RPMI 1640 MediumRPMI is a general purpose media <strong>with</strong> a broad range ofapplications for mammalian cells, especially hematopoieticcells. RPMI <strong>with</strong> MOPS (04-525F) can be used <strong>with</strong> certainmycological assays.2°C–8°C unless otherwise noted in the orderinginformation2WithL-glutamineWithoutPhenol RedWith HEPESPowderWith Penicillin–StreptomycinWithoutNa-bicarbonateWith MOPSBufferWith Ultra-Glutamine IWithoutD-glucoseMedia, Reagents and Sera / BioWhittaker Classical MediaCat. No.Size12-702F 500 ml ■12-702Q 1 l ■BE12-702F/U1 500 ml ■15-702D 1 × 10 l ■ ■ ■12-167F 500 ml12-167Q 1 lBE12-115E 100 ml ■ ■12-115F 500 ml ■ ■12-115Q 1 l ■ ■04-525F 500 ml ■ ■ ■12-918F 500 ml ■09-774E 100 ml ■ ■ ■09-774F 500 ml ■ ■ ■BE12-752F 500 ml ■ ■Ordering Information – RPMI 1640 MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE12-115E RPMI 1640 Medium With L-glutamine and 25mM HEPES 2°C–8°C 100 mlBE12-702F/U1 RPMI 1640 Medium, Powder With UltraGlutamine I 15°C–30°C 500 ml04-525F 04-525F RPMI 1640 Medium For certain mycological assays. With L-glutamine and 165 mM 2°C–8°C500 mlMOPS, <strong>with</strong>out sodium bicarbonate08-012B 08-012B RPMI 1640 Medium Without L-glutamine. Provided in exible packaging <strong>with</strong> 1 female 15°C–30°C10 land 1 male luer connector.09-774E 09-774E RPMI 1640 Medium With L-glutamine, 25 mM HEPES, 100 units/ml penicillin, and 50 2°C–8°C100 mlμg/ml streptomycin09-774F 09-774F RPMI 1640 Medium With L-glutamine, 25 mM HEPES, 100 units/ml penicillin, and 50 2°C–8°C500 mlμg/ml streptomycin12-115F BE12-115F RPMI 1640 Medium With L-glutamine and 25mM HEPES 2°C–8°C 500 ml12-115Q 12-115Q RPMI 1640 Medium With L-glutamine and 25mM HEPES 2°C–8°C 1 l12-167F BE12-167F RPMI 1640 Medium Without L-glutamine 15°C–30°C 500 ml12-167Q 12-167Q RPMI 1640 Medium Without L-glutamine 15°C–30°C 1 l12-702F BE12-702F RPMI 1640 Medium, Powder With L-glutamine 2°C–8°C 500 ml12-702Q 12-702Q RPMI 1640 Medium, Powder With L-glutamine 2°C–8°C 1 l12-918F BE12-918F RPMI 1640 Medium Without L-glutamine or phenol red 15°C–30°C 500 ml15-702D 15-702D RPMI 1640 Medium, Powder With L-glutamine 2°C–8°C 10 lBE12-752F BE12-752F RPMI 1640 Medium With L-glutamine, <strong>with</strong>out D-glucose 2°C–8°C 500 ml110North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Insect Medium2°C–8°COrdering Information – Schneider’s Drosophila MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-351Q 04-351Q Schneider‘s Drosophila Medium Modified, <strong>with</strong> L-glutamine 2°C–8°C 1 lBE02-011F TC 100 Insect Medium With L-glutamine 2°C–8°C 500 ml2Related ProductsGrace‘s Insect Medium 105Insect-XPRESS Protein-free Insect Cell Medium 133Waymouth’s Medium2°C–8°COrdering Information – Waymouth’s MB 752/1 MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE12-738F BE12-738F Waymouth‘s MB 752/1 Medium With L-glutamine 2°C–8°C 500 mlWilliam’s Medium2°C–8°COrdering Information – William’s Medium ECat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-019F BE02-019F William‘s Medium E Without L-glutamine or phenol red 2°C–8°C 500 mlBE12-761F William‘s Medium E Without L-glutamine 2°C–8°C 500 mlPageMedia, Reagents and Sera / BioWhittaker Classical MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com111
Notes2Media, Reagents and Sera / BioWhittaker Classical Media112North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
BioWhittaker Specialty Media2BioWhittaker Specialty MediaIntroduction114TheraPEAK MSCGM-CD Mesenchymal Stem CellGrowth Medium – Chemically-Defined 115FGM-CD Fibroblast Growth Medium –Chemically Defined 116UltraCULTURE Serum-free Medium 117PC-1 Chemically Defined, Serum-free Medium –Chemically Defined 118UltraMEM Reduced Serum Media 119X-VIVO Serum-free Hematopoietic Cell Media –Chemically Defined 120UltraCHO Serum-free CHO Cell Media 121ProCHO Protein-free CHO Media 122PowerCHO Serum-free CHO Media –Chemically Defined 123ProFreeze-CDM, NAO Freeze Medium –Chemically Defined 124UltraMDCK Serum-free Renal Cell Medium –Chemically Defined 124Pro293 Serum-free Media – Chemically Defined 125ProVero 1 Serum-free Medium 125ProPer 1 Serum-free Medium – Chemically Defined 126PERMEXCIS® Virus Production Medium –Chemically Defined 127Lymphochrome Medium 127Amniochrome II Modified Medium 128Amniochrome Plus Medium 128Amniochrome Pro Medium 128HL-1 Serum-free Media – Chemically Defined 129UltraDOMA Serum-free Hybridoma Medium 131ProDoma Serum-free Hybridoma Media –Chemically Defined 132UltraDOMA-PF Protein-free Hybridoma Media 132Media, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com113
Introduction2Media, Reagents and Sera / BioWhittaker Specialty MediaSerum-free media and reagents have a wide range ofapplications, including production of monoclonal antibodies,viral antigens, and recombinant proteins using a variety ofmammalian and invertebrate cell lines. There are numerousadvantages associated <strong>with</strong> the use of serum-free mediaformulations.■■Benefits––Increased definition––Increased lot-to-lot consistency––Simplified purification and downstream processing––Better control over the physiological condition ofcultures––Ability to optimize formulations for specific cell typesSerum-free media formulations must satisfy a number ofnutritional and physical requirements of cells that arenormally addressed by the presence of serum. Serumproteins, such as albumin, fibronectin, and fetuin serve avariety of functions that include adsorbing toxic compounds,providing protection against shear forces in bioreactors,creating a matrix for cellular attachment to surfaces, andacting as a carrier for lipids and other growth factors.For adaptation of cell cultures to serum-free media, seepage 431.114North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
TheraPEAK MSCGM-CD Mesenchymal Stem Cell Growth Medium –Chemically-DefinedTheraPEAK MSCGM-CD Mesenchymal Stem Cell GrowthMedium – Chemically Defined (<strong>with</strong> L-glutamine, <strong>with</strong>outphenol red and antibiotics).■■Benefits––Supports human mesenchymal stem cell growth(multi-potent adult stem cells)––Differentiation into osteoblasts, chondrocytes andadipocytes––Chemically defined and serum-free––For further manufacturing or laboratory useBasal media: 2°C–8°CSingleQuots Supplement:-10°C–-20°CRelated ProductsPoietics hMSC grown inMSCGM-CD MediumPoietics hMSC grown in DMEM<strong>with</strong> 10% FBSOrdering Information – MSCGM-CD Mesenchymal Stem Cell Basal Medium – Chemically-DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size00190620 00190620 MSCGM-CD Mesenchymal Stem Cell Basal Medium – Chemically-Defined 2°C–8°C 500 ml00190632 00190632 MSCGM-CD Mesenchymal Stem Cell Growth Medium BulletKit 15°C–30°C Kit00192125 00192125 MSCGM-CD Mesenchymal Stem Cell Growth Medium SingleQuots Supplementsand Growth Factors-10°C to -20°C KitProFreeze-CD Freeze Medium – Chemically Defined (2X) 124hMSC – Human Mesenchymal Stem Cells 87Page2Media, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com115
FGM-CD Fibroblast Growth Medium – Chemically Defined2Media, Reagents and Sera / BioWhittaker Specialty Media■■Benefits––Serum-free and chemically defined medium forexpansion––Adult – normal primary human fibroblasts––Neonatal-normal primary human fibroblasts––Maintain the fibroblasts characteristics––Promote collagen production––TheraPEAK brand help in transitioning form <strong>Research</strong>to Therapeutic applicationsBasal media: 2°C–8°CSingle Quots Supplements: -10°C– -20°CCollagen Production by NHDF-Ad grown in FGM-CDCollagen (µg)14012010080604020FibroblastAb 5B5CD900Collagen IDonor lFGM-CDFGM-CDDonor llDMEM + 10%FBSDonor lllDonor lVDMEM+10%FBSOrdering Information – FBM-CD Fibroblast Basal Medium – Chemically-DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size00199019 00199019 FBM-CD Fibroblast Basal Medium – Chemically-Defined 2°C–8°C 500 ml00199020 00199020 FGM-CD Fibroblast Growth Medium SingleQuots-10°C to -20°C KitSupplements and Growth Factors00199041 00199041 FGM-CD Fibroblast Growth Medium BulletKit Includes basal medium and SingleQuots Kit 2°C–8°C Kit116North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
UltraCULTURE Serum-free MediumGeneral Purpose Serum-free MediumUltraCULTURE Serum-free Medium is a complete,all-purpose medium designed for the cultivation of a widevariety of adherent and non-adherent mammalian celltypes. UltraCULTURE Medium can be used to support fusionof cells during hybridoma formation, growth of monocyte,macrophage, epithelial, and fibroblastic cell lines, andgeneration of virus particles for vaccine production. Themedium is supplemented <strong>with</strong> recombinant human insulin,bovine transferrin, and a purified mixture of bovine serumproteins, including albumin. The total protein concentrationof UltraCULTURE Medium is approximately 3 mg/ml.UltraCULTURE Medium can be supplemented <strong>with</strong>Cryoprotective Medium (Cat. No. 12-132A) to cryopreservecells in a serum-free environment. UltraCULTURE Mediumdoes not contain L-glutamine; please add 5 ml of 200 mML-glutamine solution (Cat. No. 17-605 or 17-905) prior touse.The formulation for UltraCULTURE Medium has beensubmitted to the FDA as a Master File. Permission to crossreferencethe Master File may be obtained by contacting theRegulatory Affairs Department.■■Applications––Cultivation of adherent and non-adherent mammaliancells––Generation of viral particles for vaccine production■■Performance and Quality Tests––Turbidity may develop in UltraCULTURE Medium;experiments have determined that the turbidity will notalter the performance of the product2°C–8°CPartial List of Cell Cultures Cultivated <strong>with</strong>UltraCULTURE MediumCell Line Source Cell TypeHEL, N-10 Human Fetal lung diploid fibroblastHeLa Human Uterine cancerHuL-1,2 Human Liver (normal)HuK-1 Human Kidney (normal)HuS-1AT Human SkinHEC Human Embryonic cancerHL-60 Human Acute promyelocytic leukemiaRaji Human Burkitt’s lymphomaEB-3 Human Burkitt’s lymphomaK-562 Human Chronic myelocytic leukemiaHNK Human Neonatal kidney (primary)HTC29 Human Colon cancerTT Human Medullary thyroid tumorMB231 Human Breast carcinomaU138 Human GliomaFM3A Mouse Breast cancerNS-1 Mouse MyelomaL Mouse SubcutaneousP388D1 Mouse Macrophage-likeP815 Mouse Mast cell tumorT3 Mouse PituitaryB82 Mouse L cells – connective tissueRPL-1 Rat PeritoneumRSP-2 Rat SpleenRLG-1 Rat LungLym-1 Rat Lymph nodeRCR-1 Rat Brain235-1, MMQ Rat PituitaryGC, GH3 Rat PituitaryCA77 Rat Medulary thyroid tumorRat-1 Rat FibroblastJTC-12 Monkey KidneyCOS1, COS7African greenmonkeySV40 transformed kidney2Media, Reagents and Sera / BioWhittaker Specialty MediaOrdering Information – UltraCULTURE Serum-free Medium, general purposeCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-725F BE12-725F UltraCULTURE Serum-free Medium, general purpose Without L-glutamine 2°C–8°C 500 mlRelated ProductsPageL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com117
PC-1 Chemically Defined, Serum-free Medium – Chemically DefinedGeneral Purpose Serum-free Medium2Media, Reagents and Sera / BioWhittaker Specialty MediaPC-1 Chemically Defined, Serum-free Medium is alow-protein medium intended for the culture of primarycells and anchorage-dependent cell lines. PC-1 Mediumcontains a complete HEPES buffering system <strong>with</strong> knownamounts of insulin, transferrin, fatty acids, and proprietaryproteins assembled under strict quality control procedures.PC-1 Medium is intended for a variety of research andindustrial applications and is formulated using definedcomponents for optimal cell growth, while maintaining thelowest possible protein content.■■Applications––Cultivation of primary and anchorage-dependent cells2°C–8°CPartial List of Cell Cultures Cultivated <strong>with</strong> PC-1Chemically Defined, Serum-free MediumCell Line Source Cell TypeHeLa Human Epithelial carcinoma, cervixHTB-72 Human Malignant melanoma, epithelial-likeHTB-4 Human Bladder tumorWISH Human Amnion, epithelial-likeWI-38 Human Lung, diploidHEP2 Human Transformed larynx, epidermoidcarcinomaMRC-5 Human, male Embryonal lung, fibroblast-likeBHK-21 Syrian hamster Kidney, fibroblast-likeCHO-K1 Chinese hamster Ovary, epithelial-likeNRK Rat Normal kidney, epithelial/fibroblastlikeC6 Rat Glioma, primaryT9 Rat GliomaARL6T Rat Normal liver3T3 Mouse Embryonic, fibroblast-likeSTO Mouse Transformed fibroblastVERO African green monkey FibroblastMDCK Dog Madin Darby canine kidney,epithelial-likeSIRC Rabbit CorneaOther Cell Types/SourcesSourceHuman:Rat:Baboon:Swine:Bovine:Cell TypeNeuroblastoma, foreskin fibroblast, bladder carcinoma,renal papillary collecting tubule (primary), colon epithelium (primary), colon carcinoma (primary)Dermal fibroblast (primary), mammary carcinoma(primary), neonatal normal cardiac muscle (primary),thyroid epithelium (primary), astrocytes (primary)(Paprocynocephalus) spinal gangliaTestes cellKidneyOrdering Information – PC-1 Serum-free Medium for Primary Adherent Cells – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size77232 77232 PC-1 Serum-free Medium for Primary AdherentCells – Chemically DefinedComplete medium system, including frozensupplement, <strong>with</strong>out L-glutamine2°C–8°C2 × 500 mlRelated ProductsPageL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124118North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
UltraMEM Reduced Serum MediaGeneral Purpose Serum-free MediaUltraMEM Reduced Serum Media are chemically definedmedia designed to support growth and maintenance ofseveral anchorage-dependent cell types under reducedserum concentrations (see table). When supplemented<strong>with</strong> 2–4% serum, UltraMEM Media growth performance iscomparable and, in some instances, exceeds that ofstandard media supplemented <strong>with</strong> 10% fetal bovine serum.Weaning is not necessary for most applications. Furtherreduction in serum concentration (
X-VIVO Serum-free Hematopoietic Cell Media – Chemically DefinedSerum-free Media for Hematopoietic Cells2Media, Reagents and Sera / BioWhittaker Specialty MediaX-VIVO Serum-free Hematopoietic Cell Media – ChemicallyDefined provide nutritionally complete and balancedenvironments for a variety of cells including lymphokineactivated killer (LAK) cells, peripheral blood lymphocytes(PBL), and tumor infiltrating lymphocytes (TIL). Thesemedia do not contain any exogenous growth factors,artificial stimulators of cellular proliferation, or undefinedsupplements. They are devoid of any protein kinase Cstimulators and are suitable for the investigation of secondmessenger systems in the activation of human and murinelymphocytes. The formulations are complete and containpharmaceutical grade human albumin, recombinant humaninsulin, and pasteurized human transferrin.All X-VIVO Media products are manufactured under currentGMPs and are listed <strong>with</strong> the FDA in a product Master File.Permission to cross-reference the Master File may be obtainedby contacting the Regulatory Affairs Department.X-VIVO 10 Serum-free Hematopoietic Cell Media –Chemically DefinedThe X-VIVO 10 Media formulations are designed tosupport the generation of LAK cells in a serum-freeenvironment. The original protocols involved the incubationof patient or normal donor peripheral blood lymphocytes(PBL) at 1.0–3.0 × 10 6 cells/ml for a period of 3 days in thepresence of 1,000 Cetus units of rIL-2/ml. Optimal LAK cellgeneration is achieved when peri pheral blood lymphocytesare incubated for 3–10 days at a density of 1.0–6.0 × 10 6cells/ml in the presence of 100–1,000 Cetus units of rIL-2.X-VIVO 10 Media is available as a 1X liquid in two convenientformulations.X-VIVO 15 Serum-free Hematopoietic Cell Media –Chemically DefinedX-VIVO 15 Media are similar in composition to X-VIVO 10 Mediaand have been optimized for the proliferation of tumorinfiltrating lymphocytes (TIL) under serum-free conditions.X-VIVO 15 Media supports the proliferation of purified CD3 +cells isolated from peripheral blood and human tumors.X-VIVO 15 Media can also be used to support the growth ofhuman monocytes, macrophage cells and cell lines, PBL,granulocytes, and natural killer (NK) cells. In addition,X-VIVO 15 Media provide a serum-free environment for theexpansion of HUT-78 and related human lymphocytic celllines.X-VIVO 20 Serum-free Hematopoietic Cell Medium –Chemically DefinedX-VIVO 20 Medium is optimized to support the generationof lymphokine activated killer (LAK) cells from monocytedepletedperipheral blood lymphocytes (PBL) at highdensity. Initial cell densities between 2.0–3.0 × 10 7 cells/ml can be used to successfully generate LAK cells. X-VIVO20 Medium may also be used as a growth medium for PBLand tumor infiltrating lymphocytes (TIL).■■Applications––Proliferation of peripheral blood lymphocytes––Proliferation of tumor infiltrating lymphocytes––Cryopreservation and transplantation of organs––Cultivation of human monocytes and macrophages––Cultivation of stem cells––Cultivation of dendritic cells2°C–8°Cwww.lonza.com/xvivoOrdering Information – X-VIVO Chemically Defined, Serum-free Hematopoietic Cell MediaCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-743Q BE04-743Q X-VIVO 10 Serum-free Hematopoietic Cell Medium –Chemically Defined04-380Q BE04-380Q X-VIVO 10 Serum-free Hematopoietic Cell Medium –Chemically Defined04-418F BE04-418F X-VIVO 15 Serum-free Hematopoietic Cell Medium –Chemically Defined04-418Q BE04-418Q X-VIVO 15 Serum-free Hematopoietic Cell Medium –Chemically Defined04-744Q X-VIVO 15 Serum-free Hematopoietic Cell Medium –Chemically Defined04-448Q BE04-448Q X-VIVO 20 Serum-free Hematopoietic Cell Medium –Chemically DefinedWith L-glutamine, <strong>with</strong>out gentamicin or 2°C–8°C1 lphenol redWith L-glutamine, gentamicin, and phenol red 2°C–8°C 1 lWith L-glutamine, gentamicin, and phenol red 2°C–8°C 500 mlWith L-glutamine, gentamicin, and phenol red 2°C–8°C 1 lWith L-glutamine, <strong>with</strong>out gentamicin or 2°C–8°C1 lphenol redWith L-glutamine, gentamicin and phenol red 2°C–8°C 1 l120North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
X-VIVO Serum-free Hematopoietic Cell Media – Chemically DefinedContinuedOrdering Information – X-VIVO 15 Serum-free Hematopoietic Cell Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-053Q BE02-053Q X-VIVO 15 Serum-free Hematopoietic Cell Medium –Chemically DefinedBE02-054Q BE02-054Q X-VIVO 15 Serum-free Hematopoietic Cell Medium –Chemically DefinedBE02-055Q BE02-055Q X-VIVO 10 Serum-free Hematopoietic Cell Medium –Chemically DefinedWith L-glutamine, gentamicin, recombinanttransferrin, and phenol redWith L-glutamine and recombinanttransferrin, <strong>with</strong>out gentamicin or phenol redWith recombinant transferrin, <strong>with</strong>outgentamicin or phenol red2°C–8°C2°C–8°C2°C–8°C1 l1 l1 l2UltraCHO Serum-free CHO Cell MediaCHO Expression MediaUltraCHO Serum-free CHO Cell Media are optimized tosupport the growth of transfected and non-transfectedChinese Hamster Ovary (CHO) cells and expression ofrecombinant proteins. UltraCHO Media are composed of amodified DMEM:F-12 base. The formulation is supplemented<strong>with</strong> insulin, transferrin, and proprietary purified proteins.UltraCHO Media are available as a complete 1X liquid or asa powder <strong>with</strong> a frozen supplement. UltraCHO Media containsless than 300 µg/ml protein.UltraCHO Media are manufactured under current GMPs andthe formulation has been submitted to the FDA as a Master File.Permission to cross-reference the file may be obtained from theRegulatory Affairs Department.■■Applications––Growth of CHO cells––Recombinant protein production■■Partial list of cell types cultivated <strong>with</strong> UltraCHOMedium––Transfected and non-transfected CHO cell lines––HeLa cells (suspension or attached)––Human leukemia cell linesLiquid medium: 2°C–8°CPowdered medium: 2°C–8°CSupplement: – 10°C–-20°COrdering Information – UltraCHO Serum-free CHO MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-724Q 12-724Q UltraCHO Serum-free CHO Medium 1X liquid <strong>with</strong> L-glutamine 2°C–8°C 1 l15-724D UltraCHO Serum-free CHO Cell Medium, Powder 2°C–8°C 10 l15-724F UltraCHO Serum-free CHO Cell Medium, Powder 2°C–8°C 50 l17-811A UltraCHO Supplement Non-sterile, for use <strong>with</strong> UltraCHO PowderedMedium10°C to -20°C 100 mlMedia, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com121
ProCHO Protein-free CHO MediaNon-animal Origin CHO Expression Media2ProCHO Protein-free CHO Media were developed specificallyto facilitate the production and downstream processing ofrecombinant proteins expressed in CHO cells. These proteinfreeformulations support high-density cultures <strong>with</strong>out theneed for animal derived components. Very low levels ofrecombinant insulin facilitate both downstream purificationand regulatory compliance. The following media systemsare available:ProMEDIA Select CHO Media Optimization KitKit contains 16 distinct formulas that contain no materialsof animal origin. It can be used to develop the mostoptimized formulas for protein expression in CHO cells.Media formulations are scalable to production volumes.■■Applications––Recombinant protein expression in CHO cellsMedia, Reagents and Sera / BioWhittaker Specialty Media––ProCHO 4 Medium – For concurrent transition ofadherent CHO cells to serum-free and suspensionculture; supports faster doubling times––ProCHO 5 Medium – For CHO cells already growingin suspension; supports increased protein production––ProCHO AT Medium – For adherent culture ofCHO cells2°C–8°COrdering Information – CHO Cell MediaCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-919Q 04-919Q ProCHO 4 Protein-free CHO Medium With 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine, phenolred, hypoxanthine or thymidine12-029Q BE12-029Q ProCHO 4 Protein-free CHO Medium With 0,1% Pluronic® F68 and phenol red, <strong>with</strong>outL-glutamine, hypoxanthine or thymidineBE02-041Q ProCHO 5 Protein-free CHO Medium With 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine, phenolred, hypoxanthine, thymidine or glucose12-766Q BE12-766Q ProCHO 5 Protein-free CHO Medium With 0.1% Pluronic® F-68, <strong>with</strong>out , L-glutamine,phenol red, hypoxanthine or thymidineBE15-766D BE15-766D ProCHO 5 Protein-free CHO Medium, Powder(1X)BE15-766F BE15-766F ProCHO 5 Protein-free CHO Medium, Powder(1X)BE02-016Q BE02-016Q ProCHO AT Serum-free Medium for AdherentCHO CellsBE18-002QProMEDIA Select CHO Media Optimization Kit<strong>with</strong>out L-glutamine, hypoxanthine, or thymidine2°C–8°C2°C–8°C2°C–8°C2°C–8°CWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine, phenolred, hypoxanthine or thymidine2°C–8°C10 lWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine, phenol 2°C–8°C50 lred, hypoxanthine or thymidineWith L-glutamine, <strong>with</strong>out hypoxanthine or thymidine 2°C–8°C 1 lKit, 16 × 1 l KitRelated ProductsPageProFreeze-CD Freeze Medium – Chemically Defined (2X) 124ProHT Supplement (100X) 138Glucose Solution 1381 l1 l1 l1 l122North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PowerCHO Serum-free CHO Media – Chemically DefinedNon-animal Origin CHO Expression MediaPowerCHO 1, 2, and 3 Chemically Defined, Serum-free CHOMedia are the next generation in CHO media, optimized forboth cell growth and protein production. PowerCHO Mediaare hydrolysate-free, serum-free, and non-animal originmedia for supporting high-density CHO cells in suspension.For therapeutic bioprocessing applications, these proteinfreeformulations also facilitate both downstreampurification and regulatory compliance.PowerCHO-GS is designed for use <strong>with</strong> Lonza’s proprietaryGS Gene Expression System.www.lonza.com/gssystemPowerCHO Media and ProCHO Media IgG Production2■■Benefits––Maximum culture performance through balancedformulation––Maintain high viability (>90%) at high cell densities––Confidence in performance lot-to-lot <strong>with</strong> chemicallydefined, serum-free media––Easily scaleable to support high-density, large scaleproduction volumes■■Application––Recombinant protein expression in CHO cells2°C–8°COrdering Information – CHO Cell MediaCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-770Q BE12-770Q PowerCHO 1 Serum-free Medium – ChemicallyDefinedBE02-042Q PowerCHO 2 Serum-free CHO Medium –Chemically DefinedBE15-771D BE15-771D PowerCHO 2 Serum-free CHO Medium –Chemically Defined, PowderBE15-771J BE15-771J PowerCHO 2 Serum-free CHO Medium –Chemically Defined, Powder12-771Q BE12-771Q PowerCHO 2 Serum-free Medium – ChemicallyDefined12-772Q BE12-772Q PowerCHO 3 Serum-free Medium – ChemicallyDefinedBE12-776Q BE12-776Q PowerCHO GS Serum-free CHO Medium –Chemically DefinedBE15-776D BE15-776D PowerCHO-GS 2 Serum-free Medium CHOMedium, PowderBE15-776F BE15-776F PowerCHO-GS 2 Serum-free Medium CHOMedium, PowderlgG (ng/ml2,5002,0001,5001,0005000ProCHO 4PowerCHO 1PowerCHO 2PowerCHO 3Commercialsupplier ACommercialsupplier BWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cphenol red, hypoxanthine or thymidineWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cphenol red, hypoxanthine, glucose or thymidineWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cphenol red, hypoxanthine or thymidineIncludes base powder and required supplements 2°C–8°C<strong>with</strong> 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine, phenolred, hypoxanthine or thymidineWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cphenol red, hypoxanthine or thymidineWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cphenol red, hypoxanthine or thymidineWith HEPES and Pluronic® F-68, <strong>with</strong>out L-glutamine, 2°C–8°Cinsulin or phenol red2°C–8°C2°C–8°CCommercialsupplier CCommercialsupplier D1 l1 l10 l100 l1 l1 l1 l10 l50 lMedia, Reagents and Sera / BioWhittaker Specialty MediaRelated ProductsPageL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124Glucose Solution 138Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com123
ProFreeze-CDM, NAO Freeze Medium – Chemically Defined (2X)Non-animal Origin Freezing Medium2Media, Reagents and Sera / BioWhittaker Specialty MediaProFreeze- CDM, NAO Freeze Medium – Chemically Defined(2X) is universally suitable for cryopreserving many celltypes in the absence of fetal bovine serum (FBS). However,it is used to greatest advantage <strong>with</strong> cells cultured in aserum-free and animal component-free environment. Thisprotein-free freezing medium contains no animal derivedcomponents, insulin, or hydrolysate, and maintains highcell viability upon recovery from frozen storage.ProFreeze Medium requires the addition of 15% reagent orspectrophotometric grade dimethylsulfoxide (DMSO) attime of use. One bottle will make 117.6 ml of complete 2Xconcentrated freezing medium after the addition of 17.6 mlDMSO. For best results, keep on ice during use.UltraMDCK Serum-free Renal Cell Medium – Chemically DefinedRenal Cell Expression MediumUltraMDCK Serum-free Renal Cell Medium – ChemicallyDefined is a defined, serum-free medium designed tosupport the growth of MADIN-DARBY Canine Kidney (MDCK)cells at low and high plating densities. UltraMDCK Mediumcontains low levels of recombinant human insulin andbovine transferrin, yielding a very low protein formulation.MDCK cells grown in UltraMDCK Medium are smaller andmore densely packed than cells grown in the presence ofserum, and cultures can stay confluent for at least twoweeks <strong>with</strong>out medium change.2°C–8°COrdering Information – ProFreeze-CD Freeze Medium – Chemically Defined (2X)Cat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-769E 12-769E ProFreeze-CD Freeze Medium – Chemically Defined (2X) Non-Animal Origin 2°C–8°C 100 ml■■Applications––Growth of MDCK cells2°C–8°COrdering Information – UltraMDCK Serum-free Renal Cell Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-749Q BE12-749Q UltraMDCK Serum-free Renal Cell Medium – Chemically Defined With L-glutamine 2°C–8°C 1 l124North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pro293 Serum-free Media – Chemically DefinedNon-animal Origin Renal Cell Expression MediaPro293 Serum-free Media – Chemically Defined wereoptimized to support high-density growth and recombinantprotein production in 293 neonatal kidney cells. They arechemically defined to ease regulatory compliance anddownstream protein purification. They contain very lowlevels of recombinant human insulin, and are free of animalorigincomponents.––Pro293s Medium – for 293 cells growing in suspensionculture––Pro293 a Medium – for 293 cells growing in adherentcultureProVero 1 Serum-free MediumNon-animal Origin Renal Cell Expression MediumProVero 1 Serum-free Medium is a protein-free mediumdesigned to support the growth of MDCK and Vero cells.ProVero 1 Medium includes HEPES and sodium bicarbonatebuffer. The absence of proteins and only very low levels ofhuman recombinant insulin facilitate both downstreamprocessing and regulatory compliance.■■Applications––Recombinant protein production in 293 cells2°C–8°COrdering Information – Pro293s Serum-free Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-765Q 12-765Q Pro293s Serum-free Medium – Chemically Defined for293 Cells in SuspensionBE02-025Q Pro293s Serum-free Medium – Chemically Defined for293 Cells in Suspension12-764Q BE12-764Q Pro293a Serum-free Medium – Chemically Defined for293 Adherent CellsRelated ProductsWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamineor phenol redWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamineor phenol redWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamineor phenol red■■Applications––Recombinant protein production––Virus production2°C–8°C2°C–8°C2°C–8°C2°C–8°CL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 1241 l1 l1 lPage2Media, Reagents and Sera / BioWhittaker Specialty MediaSome Vero cell strains require additional supplementation <strong>with</strong>5.0 µg/L rhEGF for optimal vero cell growth.Ordering Information – ProVero 1 Serum-free MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-030Q BE02-030Q ProVero 1 Serum-free Medium With L-glutamine 2°C–8°C 1 lRelated ProductsProFreeze-CD Freeze Medium – Chemically Defined (2X) 124PageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com125
ProPer 1 Serum-free Medium – Chemically DefinedNon-animal Origin Medium for Human Embryonic Retinoblast Cells2ProPer 1 Serum-free Medium – Chemically Defined is ananimal origin component-free, chemically defined, serumfreemedium for growth of human embryonic retinoblastcells (PER.C6® and related cell lines) in suspension. Themedium contains a low amount of human recombinantinsulin. HEPES as well as sodium bicarbonate are present inthe formulation.Media, Reagents and Sera / BioWhittaker Specialty Media■■Applications––Recombinant protein and virus production––Growth of human embryonic retinoblast cells(PER.C6® and related cell lines) in suspension2°C–8°CRelated ProductsPER.C6® Cells growing in ProPer 1 Medium.Ordering Information – ProPer 1 Serum-free Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-028Q BE02-028Q ProPer 1 Serum-free Medium – ChemicallyDefinedWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine orphenol red2°C–8°CL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 1241 lPage126North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PERMEXCIS® Virus Production Medium – Chemically DefinedNon-animal Origin Medium for Human Embryonic Retinoblast CellsPER.C6® Technology is a human cell-based platformdesigned for the large-scale production of recombinantproteins, including antibodies, vaccines and gene therapyproducts, under serum-free culture conditions. Cell lineshave been immortalized <strong>with</strong> specific adenovector genes,leading to high viable cell densities and high PCDs(picogram/cell/day), resulting in high yields.PERMEXCIS® Virus Production Medium has been optimizedfor use <strong>with</strong> the PER.C6® Cell Line and is available to allPER.C6® Cell License Holders. PERMEXCIS® Medium ischemically defined, serum-free, low protein (
Amniochrome II Modified MediumCytogenetics Medium2Amniochrome II Modified Medium is for the culture ofhuman amniotic fluid cells obtained from amniocentesis, aprocedure extensively used for clinical prenatal diagnosis.The Amniochrome II Modified Medium is performance testedby a cytogenetic reference laboratory for the cultivation ofamniotic fluid cells for cytogenetic analysis.Complete medium for the primary culture of amniotic fluidand chorionic villi cells used in cytogenetic applications. Thesystem includes an enriched basal medium and growthsupplement. For diagnostic use.Basal medium: 2°C–8°CSupplement:-10°C–-20°CMedia, Reagents and Sera / BioWhittaker Specialty MediaOrdering Information – Amniochrome II Modified MediumCat. No. NA Cat. No. EU Product Name Storage Conditions SizeBE12-756EZM Amniochrome II Modified Medium Basal medium: 2°C–8°C, Supplement: – 10°C – – 20°C 100 mlBE12-756FCM Amniochrome II Modified Medium Basal medium: 2°C–8°C, Supplement: – 10°C – – 20°C 500 mlCE marked according to IVD Directive 98/79/EC.Amniochrome Plus MediumCytogenetics MediumComplete, ready-to-use medium for the primary culture ofamniotic fluid and chorionic villi cells used in cytogeneticapplications. Quick attachment of the cells and high growthspeed of cells are the main advantages of this new mediaformulation. For in vitro diagnostic use.Amniochrome Pro MediumCytogenetics MediumComplete, ready-to-use medium for the primary culture ofamniotic fluid and chorionic villi cells used in cytogeneticapplications. Contains the necessary growth factors,L-glutamine, phenol red, sodium bicarbonate, and FBS. Thecomplete formulation reduces handling steps and thepossibility of contamination. For diagnostic use.-10°C–-20°COrdering Information – Amniochrome Plus MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-026E Amniochrome Plus Medium -10°C to -20°C 100 mlBE02-026F Amniochrome Plus Medium -10°C to -20°C 500 mlCE marked according to IVD Directive 98/79/EC.-10°C–-20°COrdering Information – Amniochrome Pro MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-035E Amniochrome Pro Medium -10°C to -20°C 100 mlBE02-035F Amniochrome Pro Medium -10°C to -20°C 500 mlCE marked according to IVD Directive 98/79/EC.128North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
HL-1 Serum-free Media – Chemically DefinedChemically Defined Hybridoma MediumHL-1 Serum-free Media – Chemically Defined is a culturemedium containing less than 30 µg protein per ml.Components of HL-1 Medium include HEPES buffer, insulin,transferrin, sodium selenite, ethanolamine, a variety ofsaturated and unsaturated fatty acids and proprietarystabilizing proteins. HL-1 Medium contains no bovineserum albumin or other undefined protein mixtures. HL-1Medium supports the serum-free growth of varioushybridomas, including those derived from P3X63Ag8.653and Sp2/0-Ag14 myelomas, as well as other differentiatedcells of lymphoid origin.■■Applications––Serum-free growth of hybridomas and differentiatedcells of lymphoid origin2°C–8°CRelated ProductsHL-1 FBS Substitute – Chemically Defined (100X)HL-1 Chemically Defined FBS Substitute (100X) is achemically defined medium additive that can be used toreplace serum or significantly reduce its concentration in avariety of basal media. It contains less than 30 µg protein/ml when diluted 1:100 in medium and it does not containbovine serum albumin or other undefined proteiningredients.15°C–30°COrdering Information – HL-1 Serum-free Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size77204 77204 HL-1 Serum-free Medium – Chemically Defined, Powder Without L-glutamine 2°C–8°C 50 l77227 77227 HL-1 Fetal Bovine Serum Substitute – Chemically Defined (100X) 15°C–30°C 10 ml77201 BE77201 HL-1 Serum-free Medium for Hybridoma and Hematopoietic Cells – 1X liquid, <strong>with</strong>out L-glutamine 2°C–8°C 2 × 500 mlChemically DefinedCE marked according to IVD Directive 98/79/EC.L-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124Page2Media, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com129
HL-1 Serum-free Media – Chemically DefinedContinuedPartial list of cell cultures cultivated <strong>with</strong> HL-1 Chemically Defined, Serum-free Medium2Media, Reagents and Sera / BioWhittaker Specialty MediaTransformed and Established Cell LinesCell Line Source Cell TypeU937 Human MacrophageRaJi Human B lymphoblasticMCF-7 (NIH) Human Breast carcinomaMCF-7 (MCF) Human Breast carcinomaNIH ZR-75 Human Breast carcinomaCOLO 302 HSR Human Colon carcinomaJ82 Human Bladder carcinomaSW 1738 Human Bladder carcinomaSW780 Human Bladder carcinomaCCL 119 Human LymphoidCCL 213 Human Burkitt lymphomaC91/PL Human T lymphomaUndesignated Human AstrocytomaUndesignated Human HepatomaMOLT-3 Human Acute lymphoblasticleukemiaMOLT-4 Human Acute lymphoblasticleukemiaNAMALWA Human Burkitt lymphomaTHP-1 Human Monocytic leukemiaBB88 Mouse Erythroid (leukemia)P815 Mouse MacrophageP388D1 Mouse MacrophageWeHi3 Mouse MonocyteJLS-V5 Mouse Spleen cell70Z-3 Mouse Pre-B lymphoma70Z/3.12 Mouse B lymphomaS49 and variants Mouse T lymphomaRAW309F1.1 Mouse T lymphomaWeHi7 Mouse T lymphomaI-10 Mouse Leydig-tumorEL4 Mouse T lymphomaRL1 Mouse T lymphomaBW5147.3 Mouse T lymphomaLBRM-33 Mouse T lymphomaFriend leukemia Mouse LeukemiaC57BL6 Mouse Embryo (C57 × DBA)L5178Y Mouse Lymphoma (DBA/2)VEROAfrican greenFibroblastmonkeyMDCK Dog Madin Darby canine kidneyCHO K1 Hamster Chinese hamster ovary(epithelial-like)GCL2 Hamster/mouse B lymphoma × Normal BHybridomasHybridoma Source Fusion Partner8A1 Human CLLCundesignated Human WI-L2-729-HF2undesignated Human LICR-LON-HMY2HB44 Mouse Sp2/0-Ag14HB45 Mouse Sp2/0-Ag14HB56 Mouse NS-1HB59 Mouse NS-1HB60 Mouse P3X63Ag 8.65353-7.313 Mouse NS-1MI/9.3.4HL-2 Mouse NS-1MI/70.15.1 Mouse NS-1ARB Mouse HybridomaP3U Mouse P3X63Ag 8.653BCS12 Mouse P3X63Ag 8.653BCS 2002 Mouse P3X63Ag 8.653undesignated Mouse NS-1undesignated Mouse P3X63Ag 8.653TIB 175 Rat/mouse S194TIB 104 Rat/mouse NS-1TIB 105 Rat/mouse NS-1TIB 109 Rat/mouse NS-1TIB 128 Rat/mouse NS-1TIB 166 Rat/mouse NS-1TIB 168 Rat/mouse NS-1RS Rat/mouse P3X63Ag 8.653Primary CellsCell TypeHuman peripheral blood mononuclear cellsMink lymphocytesHuman fetal adrenalHuman blood monocytesHuman peripheral blood T lymphocytes130North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
UltraDOMA Serum-free Hybridoma MediumHybridoma MediumUltraDOMA Serum-free Hybridoma Medium is a formulationdesigned for the cultivation of murine, human, and chimerichybridomas in batch culture and in hollow fiber bioreactors.UltraDOMA Medium is supplemented <strong>with</strong> recombinanthuman insulin, bovine transferrin and bovine albumin. Thetotal protein concentration is 30 µg/ml. UltraDOMA Mediumdoes not contain L-glutamine.Cells that are adapted for growth in UltraDOMA Medium canbe maintained in the medium indefinitely and can be cryopreservedin UltraDOMA Medium supplemented <strong>with</strong>Cryoprotective Freezing Medium (Cat. No. 12-132A).■■Applications––Hybridoma cell growth––Monoclonal antibody production■■Performance and Quality Tests––UltraDOMA Medium contains no human-derivedproteins2°C–8°CPartial List of Cell Types Cultivated <strong>with</strong> UltraDOMAMediumCell TypeMurine hybridomasNS-1 derived myelomasSP-2 derived myelomasHuman hybridomas (<strong>with</strong> 0.5% FBS)Ordering Information – UltraDOMA Serum-free Hybridoma MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-723B 12-723B UltraDOMA Serum-free Hybridoma Medium Without L-glutamine 2°C–8°C 500 ml (glass bottle)BE12-723F UltraDOMA Serum-free Hybridoma Medium Without L-glutamine 2°C–8°C 500 ml (plastic bottle)Related ProductsPageL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124UltraGlutamine 1392Media, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com131
ProDoma Serum-free Hybridoma Media – Chemically DefinedNon-animal Origin Hybridoma Media2ProDoma Serum-free Hybridoma Media is designed forcultivation of murine, human, and chimeric hybridomas.ProDoma Media are protein-free <strong>with</strong> a low amount ofhuman recombinant insulin. All ProDoma Media includeHEPES as well as sodium bicarbonate in the formulation.■■Applications––Hybridoma cell growth––Monoclonal antibody production2°C–8°CMedia, Reagents and Sera / BioWhittaker Specialty MediaOrdering Information – ProDOMA 1 Serum-free Hybridoma Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-029Q BE02-029Q ProDOMA 1 Serum-free Hybridoma Medium –Chemically DefinedBE02-032Q BE02-032Q ProDOMA 3 Serum-free Hybridoma Medium –Chemically DefinedUltraDOMA-PF Protein-free Hybridoma MediaNon-animal Origin Hybridoma MediaUltraDOMA-PF Protein-free Hybridoma Media areformulations designed for use <strong>with</strong> hybridoma cell lines ofmurine, human, and chimeric origin. UltraDOMA-PF Mediaare completely defined media and do not contain peptides ortissue extracts. The use of UltraDOMA-PF Media significantlysimplifies downstream processing since all proteins presentin a given cell culture supernatant are produced by the cells.UltraDOMA-PF Media are designed for lab scale or industrialscale use. It is available as a 1X liquid or as a powder.L-glutamine and HEPES buffer are included in theformulation.■■Applications––Hybridoma and myeloma growth––Monoclonal antibody production2°C–8°CWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine orphenol redWith 0.1% Pluronic® F-68, <strong>with</strong>out L-glutamine orphenol red2°C–8°C2°C–8°CPartial List of Cell Types Cultivated <strong>with</strong>UltraDOMA-PF MediumCell TypeMurine hybridomasNS-1 derived myelomasSP-2 derived myelomasHuman hybridomas (<strong>with</strong> 0.5% FBS)Rat hybridomasSome transfected Chinese Hamster Ovary (CHO) cell linesHuman lymphoid origin cellsMurine lymphoid origin cells1 l1 lOrdering Information – UltraDOMA Protein-free Hybridoma MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-727F 12-727F UltraDOMA Protein-free Hybridoma Medium 1X liquid, <strong>with</strong> L-glutamine 2°C–8°C 500 ml132North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Insect-XPRESS Protein-free Insect Cell MediumInsect Cell Expression MediumInsect-XPRESS Protein-free Insect Cell Medium is aformulation designed to support the growth of insect celllines derived from Spodoptera frugiperda (Sf9 and Sf21).Cell densities in excess of 8.3 × 10 6 cells/ml can be achieved<strong>with</strong> suspension cultures of Sf9 cells using Insect-XPRESSMedium and an excess of oxygen. This formulation can alsobe used for stationary mono layer cultures and shake-flaskcultures. Insect-XPRESS Medium contains L-glutamine andsupports superior production of recom binant proteins whenusing the Baculovirus Expression Vector System (BEVS).For cryopreser vation of insect cells, Insect-XPRESSMedium can be mixed 50:50 <strong>with</strong> Cryoprotective FreezeMedium (Cat. No. 12-132A).2°C–8°COrdering Information – Insect-XPRESS Protein-free Insect Cell MediumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-730F BE12-730F Insect-XPRESS Protein-free Insect Cell Medium With L-glutamine 2°C–8°C 500 ml12-730Q BE12-730Q Insect-XPRESS Protein-free Insect Cell Medium With L-glutamine 2°C–8°C 1 lRelated ProductsPageProFreeze-CD Freeze Medium – Chemically Defined (2X) 124Insect-XPRESS Protein-free Insect Cell Medium 133Schneider‘s Drosophila Medium 1112Media, Reagents and Sera / BioWhittaker Specialty MediaEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com133
ProNS0 Protein-free Media – Chemically DefinedNon-animal Origin NS0 Expression Media2Media, Reagents and Sera / BioWhittaker Specialty MediaThe NS0 cell line is widely used for recombinant mammalianprotein expression. Some of the reasons to select thismouse myeloma platform are:––Forms stably producing hybrid cells <strong>with</strong> high levels ofprotein production––Lacks ability to secrete endogenous antibody orantibody fragments––Resists aggregation clumping in suspension culture––Lacks adverse protease activity on recombinant proteinproduct (in many cases)ProNS0 1 and 2 Chemically Defined (CD) Media, together<strong>with</strong> ProNS0 Lipid CD Supplement, are designed to meet agrowing demand for optimized NS0 formulations.––Two optimized formulas are available to cover thebroadest range of NS0 nutritional needs––Further maximize protein production by titration <strong>with</strong>ProNS0 Lipid CD Supplement––Protein-free and chemically defined formulations––Product purification is simplified––Lot-to-lot consistency ensures dependableperformance––Non-animal origin components reduce regulatoryburdensData in the figure to the right shows both ProNS0 1 and2 CD Media yield superior protein production compared tocompetitive media. Other experiments displayed the sametrends (data not shown).■■Applications––High density culture of NS0 cellsBasal media: 2°C–8°CLipid supplement:-10°C–-20°CProtein Production – ProNSO MedialgG (mg/ml)0.20.150.10.050ProNSO 1 CDDay 6 mg/mlProNSO 2 CDProNS0 Media outperforms competitive media for recombinant IgGproduction in NS0 cells. Duplicate 125 ml shaker flasks were seeded at adensity of 200,000 NS0 cells per ml in a 30 ml volume. Shake rate was100 rpm. Cells were cultured in their respective test media for one passageprior to test initiation.Competitor 1Competitor 2Competitor 3Ordering Information – ProNS0 Protein-free Medium – Chemically DefinedCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size12-773Q 12-773Q ProNS0 1 Protein-free Medium – ChemicallyDefined12-774Q 12-774Q ProNS0 2 Protein-free Medium – ChemicallyDefined12-775J 12-775J ProNS0 Lipid Supplement – ChemicallyDefinedWith HEPES and Pluronic®, <strong>with</strong>out L-glutamine, phenolred or cholesterol2°C–8°C1 lWith HEPES and Pluronic®, <strong>with</strong>out L-glutamine, phenol 2°C–8°C1 lred or cholesterolWith cholesterol, suggested use is 5 ml/l -10°C to -20°C 5 mlRelated ProductsPageL-glutamine 138ProFreeze-CD Freeze Medium – Chemically Defined (2X) 124134North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
BioWhittaker Cell Culture Reagents2Insect-XPRESS Protein-free Insect Cell Medium 133ProNS0 Protein-free Media – Chemically Defined 134BioWhittaker Cell Culture ReagentsIntroduction136Balanced Salt Solutions 137Earle’s BSS 137Hanks’ BSS 137Reagents138Growth Factors 140Antibiotics and Antimycotics 140Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com135
Introduction2BioWhittaker Cell Culture Reagents include a number ofdifferent products, such as amino acids, antibiotics, andbuffers, all of which are used routinely in research, andmanufacturing applications involving cell culture.These products are manufactured under the same cGMPconditions as our other cell culture products.Chemicals we use to prepare cell culture reagents arepurchased according to the raw material qualifications fromapproved suppliers. Each lot must meet establishedcomponent specifications before it is released by QualityAssurance for use. We manufacture all liquid cell culturereagents using Water for Injection (WFI) quality water, whichhas been prepared by ultrafiltration, reverse osmosis,deionization, and distillation. Liquid products are sterilefiltered through pharmaceutical-grade sterilizing filters.Media, Reagents and Sera / BioWhittaker Cell Culture Reagents136North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Balanced Salt SolutionsEarle’s BSS15°C–30°COrdering Information – Earle’s Buffered Saline SolutionCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE10-502F Earle‘s Buffered Saline Solution With phenol red 15°C–30°C 500 mlBE02-027F Earle‘s Buffered Saline Solution With 20 mM HEPES, 1.8 g/L sodium bicarbonate and phenol red 15°C–30°C 500 ml2Hanks’ BSS (1X)Cat. No.SizeWithPhenol RedWithoutPhenol RedWithCalcium andMagnesiumWithoutCalcium orMagnesium10-508F 500 ml ■ ■10-508Q 1 l ■ ■10-543F 500 ml ■ ■10-543Q 1 l ■ ■10-527F 500 ml ■ ■10-547F 500 ml ■ ■04-315Q 1 l ■ ■08-003A 4 l (bag) ■ ■For exact compositions / product formulations, see page 439-449.15°C–30°COrdering Information – Hank’s Buffered Saline SolutionCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size04-315Q 04-315Q Hank‘s Buffered Saline Solution Without phenol red, calcium or magnesium 15°C–30°C 1 l08-003A 08-003A Hank‘s Buffered Saline Solution Without phenol red, calcium or magnesium. Provided in a flexible 15°C–30°C4 lpackage <strong>with</strong> one female luer connector, one blood spike receptacle,and one male MPC connector <strong>with</strong> cap.10-508Q 10-508Q Hank‘s Buffered Saline Solution With phenol red, calcium and magnesium 15°C–30°C 1 l10-543Q 10-543Q Hank‘s Buffered Saline Solution With phenol red, <strong>with</strong>out calcium or magnesium 15°C–30°C 1 l10-508F BE10-508F Hank‘s Buffered Saline Solution With phenol red, calcium and magnesium 15°C–30°C 500 ml10-527F BE10-527F Hank‘s Buffered Saline Solution Without phenol red, <strong>with</strong> calcium and magnesium 15°C–30°C 500 ml10-543F BE10-543F Hank‘s Buffered Saline Solution With phenol red, <strong>with</strong>out calcium or magnesium 15°C–30°C 500 ml10-547F BE10-547F Hank‘s Buffered Saline Solution Without phenol red, calcium or magnesium 15°C–30°C 500 mlMedia, Reagents and Sera / BioWhittaker Cell Culture ReagentsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com137
Reagents2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsOrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE02-040E BE02-040E Glucose Solution 100 mg/ml 15°C–30°C 100 ml17-839Z 17-839Z ITES (500X) Supplement of insulin, transferrin, selenium and-10°C to -20°C 5 mlethanolamine17-838Z 17-838Z ITS (500X) Supplement of insulin, transferrin and selenium -10°C to -20°C 5 ml17-905C 17-905C L-Glutamine, 200 mM Supplied at 29.3 mg/ml in 0.85% NaCl and hybridoma -10°C to -20°C 50 mlscreened17-605C 17-605C L-Glutamine, 200 mM Supplied at 29.3 mg/ml in 0.85% NaCl -10°C to -20°C 50 ml17-605F BE17-605F L-Glutamine, 200 mM Supplied at 29.3 mg/ml in 0.85% NaCl -10°C to -20°C 500 ml17-605E BE17-605E L-Glutamine, 200 mM Supplied at 29.3 mg/ml in 0.85% NaCl -10°C to -20°C 100 ml15-605G BE15-605G L-Glutamine, Powder 2°C–8°C 25 g17-829E 17-829E Lymphocyte Separation Medium, 1.077 Density 1.077, for the isolation of human lymphocytes 15°C–30°C 100 ml17-829F 17-829F Lymphocyte Separation Medium, 1.077 Density 1.077, for the isolation of human lymphocytes 15°C–30°C 500 ml13-607C 13-607C MEM Eagle Vitamine Mixture (100X) -20°C 50 ml13-114E BE13-114E MEM Non-Essential Amino Acid Solution Contains a 10 mM concentration of each non-essential 2°C–8°C100 ml(100X)amino acidBE17-855E BE17-855E ProHT Supplement (100X) Hypoxanthine, Thymidine supplement (100X) from -10°C to -20°C 100 mlnon-animal origin, optimized for use <strong>with</strong> ProCHOMedia (12-766Q, 12-029Q, 04-919Q, BE02-016Q)17-613E BE17-613E Sodium Bicarbonate Solution, 7.5% 7.5% aqueous solution 15°C–30°C 100 ml15-613I 15-613I Sodium Bicarbonate, Powder 15°C–30°C 500 g13-115E BE13-115E Sodium Pyruvate Solution (100 mM) 11.1 g/l 2°C–8°C 100 ml17-942E 17-942E Trypan Blue, 0.4% Solution Prepared in 0.85% NaCl 15°C–30°C 100 ml17-160F 17-160F Trypsin 1:250 (10X) 2.5% in modified Hanks’ BSS <strong>with</strong>out calcium or-10°C to -20°C 500 mlmagnesium, manufactured <strong>with</strong> irradiated porcinetrypsin, tested for porcine parvovirus and mycoplasma17-160E BE17-160E Trypsin 1:250 (10X) 2.5% in modified Hanks’ BSS <strong>with</strong>out calcium or-10°C to -20°C 100 mlmagnesium, manufactured <strong>with</strong> irradiated porcinetrypsin, tested for porcine parvovirus and mycoplasmaBE02-007E BE02-007E Trypsin/EDTA (10X) Includes 5 g/L trypsin 1:250 and 2 g/L Versene® -10°C to -20°C 100 ml(EDTA), manufactured <strong>with</strong> irradiated porcine trypsin,tested for porcine parvovirus and mycoplasma17-161E BE17-161E Trypsin/EDTA (1X) Contains 0.5 g/L trypsin 1:250 and 0.2 g/L Versene® -10°C to -20°C 100 ml(EDTA), manufactured <strong>with</strong> irradiated porcine trypsin,tested for porcine parvovirus and mycoplasma17-161F BE17-161F Trypsin/EDTA (1X) Contains 0.5 g/L trypsin 1:250 and 0.2 g/L Versene® -10°C to -20°C 500 ml(EDTA), manufactured <strong>with</strong> irradiated porcine trypsin,tested for porcine parvovirus and mycoplasmaBE02-034E Trypzean Trypsin/EDTA (10X) -10°C to -20°C 100 ml138North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ReagentsContinuedOrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeBE17-605E/U1BE17-605E/U1BE17-605E/U1/12BE04-684E/12UltraGlutamine I Supplement, 200 mM(100X)UltraGlutamine I Supplement, 200 mM(100X)UltraGlutamine II Supplement, 200mM (100X)BE04-684E UltraGlutamine II Supplement, 200mM (100X)200 mM Alanyl-L-glutamine in normal saline. Thisvery stable form of L-glutamine is used at equimolarconcentrations to L-glutamine and requires little to noadaptive period.200 mM Alanyl-L-glutamine in normal saline. Thisvery stable form of L-glutamine is used at equimolarconcentrations to L-glutamine and requires little to noadaptive period.200 mM L-Gycyl-L-glutamine in normal saline. Thisvery stable form of L-glutamine is used at equimolarconcentrations to L-glutamine and may require anadaptive period.200 mM L-Gycyl-L-glutamine in normal saline. Thisvery stable form of L-glutamine is used at equimolarconcentrations to L-glutamine and may require anadaptive period.15°C–30°C100 ml15°C–30°C 12 × 100ml15°C–30°C 12 × 100ml15°C–30°C17-724F BE17-724F Water for Cell Culture Water for injection (WFI) quality water is prepared 15°C–30°C500 mlby ultrafiltration, reverse osmosis, deionization,distillation, and sterile filtration17-724Q BE17-724Q Water for Cell Culture Water for injection (WFI) quality water is prepared 15°C–30°C1 lby ultrafiltration, reverse osmosis, deionization,distillation, and sterile filtration04-585P 04-585P Water for Cell Culture (screw-cap, carboy) 15°C–30°C 20 l08-199D 08-199D Water for Cell Culture (flexible packaging) 15°C–30°C 100 l08-199E 08-199E Water for Cell Culture (flexible packaging) 15°C–30°C 200 l08-199F 08-199F Water for Cell Culture (flexible packaging) 15°C–30°C 20 l100 ml2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com139
Growth Factors2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsOrdering Information – Growth FactorsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeCC-4398 CC-4398 Ascorbic Acid 25.5 mg/ml -10°C to -20°C 0.5 mlCC-4098 CC-4098 Bovine Brain Extract 9 mg/ml -10°C to -20°C 5 mlCC-4092 CC-4092 Bovine Brain Extract 3 mg/ml -10°C to -20°C 2 mlCC-4009 CC-4009 Bovine Pituitary Extract 13 mg/ml -10°C to -20°C 2 mlCC-4202 CC-4202 Calcium Chloride 300 mM 15°C–30°C 2 mlCC-4107 CC-4107 hEGF Human Epidermal Growth Factor 3 µg/ml -10°C to -20°C 0.5 mlCC-4068 CC-4068 hFGF – Human Fibroblastic Growth Factor 1 µg/ml -10°C to -20°C 1 mlCC-4205 CC-4205 Human Transferrin 10 mg/ml -10°C to -20°C 0.5 mlCC-4323 CC-4323 NSF-1 Neural Survival Factor-1 -10°C to -20°C 4 mlAdditional Cell Culture Reagents can be found on page73.Antibiotics and Antimycotics15°C–30°C unless otherwise noted in orderinginformationOrdering Information – Antibiotics and AntimycoticsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size17-836E 17-836E Amphotericin B Contains 250 μg/ml amphotericin B -10°C to -20°C 100 ml17-836R 17-836R Amphotericin B Contains 250 μg/ml amphotericin B -10°C to -20°C 20 ml15-394N 15-394N G418 Selective Antibiotic, Powder 2°C–8°C 5 g17-518L 17-518L Gentamicin Sulfate 50 mg/ml 15°C–30°C 10 × 10 ml (screw cap vial)17-518Z 17-518Z Gentamicin Sulfate 50 mg/ml 15°C–30°C 1 × 10 ml (screw cap vial)17-519L 17-519L Gentamicin Sulfate 10 mg/ml 15°C–30°C 10 × 10 ml (screw cap vial)17-519Z 17-519Z Gentamicin Sulfate 10 mg/ml 15°C–30°C 1 × 10 ml (screw cap vial)17-528Z 17-528Z Gentamicin Sulfate 50 mg/ml 15°C–30°C 1 × 10 ml (crimp top vial)BE02-012E Gentamicin Sulfate 10 mg/ml 15°C–30°C 100 ml140North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Penicillin-Streptomycin Mixtures5,000 UnitsPenicillin –5,000 μgStreptomycin10,000 UnitsPenicillin –10,000 μgStreptomycin20,000 UnitsPenicillin –20,000 μgStreptomycin25,000 UnitsPenicillin –25,000 μgStreptomycin25 µg/mlAmphotericinBWithL-glutamineCat. No.Size17-603E 100 ml ■17-602E 100 ml ■17-602F 500 ml ■17-718R 25 × 4.5 ml ■ ■09-757F 500 ml ■17-745H 20 ml ■ ■17-745E 100 ml ■ ■For exact compositions, see reagents formulation section, page 439–455.-10°C–-20°COrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size09-757F 09-757F Penicillin-Streptomycin Mixture Contains 20,000 units potassium penicillin and20,000 μg streptomycin sulfate per ml in 0.85%saline17-602E DE17-602E Penicillin-Streptomycin Mixture Contains 10,000 units potassium penicillin and10,000 μg streptomycin sulfate per ml in 0.85%saline17-602F DE17-602F Penicillin-Streptomycin Mixture Contains 10,000 units potassium penicillin and10,000 μg streptomycin sulfate per ml in 0.85%saline17-603E DE17-603E Penicillin-Streptomycin Mixture Contains 5,000 units potassium penicillin and5,000 μg streptomycin sulfate per ml in 0.85%saline17-745E 17-745E Penicillin-Streptomycin-Amphotericin B Mixture Contains 10,000 units potassium penicillin,10,000 μg streptomycin sulfate and 25 μgAmphotericin B per ml in 0.85% saline17-745H 17-745H Penicillin-Streptomycin-Amphotericin B Mixture Contains 10,000 units potassium penicillin,10,000 μg streptomycin sulfate and 25 μgAmphotericin B per ml in 0.85% saline17-718R 17-718R Penicillin-Streptomycin-L-Glutamine Mixture Contains 25,000 units potassium penicillin,25,000 μg streptomycin sulfate and 29.2 mgL-glutamine per mlRelated Products-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°CMycoZap Antibiotics 161MycoAlert PLUS Mycoplasma Detection Kit 157500 ml100 ml500 ml100 ml100 ml20 ml25 × 4.5 mlPage2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com141
Buffers and Buffered SalinesBuffers15°C–30°C2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsOrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size10-548E 10-548E ACK Lysing Buffer (1X) Used to lyse red blood cells in preparations containing 15°C–30°C100 mlwhite blood cells10-539B 10-539B Dextrose-Gelatin-Veronal 15°C–30°C 500 mlDulbecco’s Phosphate Buffered SalineWithoutPhenol RedWithCalcium andMagnesiumWithoutCalcium orMagnesiumWithGlucoseWithSodiumPyruvateCat. No. Size17-513F 500 ml ■ ■17-513Q 1 l ■ ■15-456D 1 × 10 l ■ ■ ■15-456F 1 × 50 l ■ ■ ■BE15-512D 1 × 10 l ■ ■ ■BE15-512F 1 × 50 l ■ ■ ■17-512F 500 ml ■ ■17-512Q 1 l ■ ■04-479Q 1 l ■ ■ ■ ■17-515F 500 ml (10X) ■ ■17-515Q 1 l (10X) ■ ■PowderFor exact compositions, see reagents formulation section, page 439–455.15°C–30°COrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size17-515F BE17-515F Dulbecco‘s Phosphate Buffered Saline (10X) 95 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 10 × 500 ml17-515Q BE17-515Q Dulbecco‘s Phosphate Buffered Saline (10X) 95 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 10 × 1 l04-479Q 04-479Q Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO 4) <strong>with</strong> calcium, magnesium, 1 g/l 15°C–30°C1 lglucose and 36 mg/l sodium pyruvate, used inanimal embryo transfer procedures17-512F BE17-512F Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 500 ml17-512Q BE17-512Q Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 1 l17-513F BE17-513F Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO 4) <strong>with</strong> calcium and 9.5 mM (PO4) 15°C–30°C500 mlmagnesium17-513Q BE17-513Q Dulbecco‘s Phosphate Buffered Saline (1X) 9.5 mM (PO 4) <strong>with</strong> calcium and 9.5 mM (PO 4) 15°C–30°C1 lmagnesium15-456D 15-456D Dulbecco‘s Phosphate Buffered Saline, Powder 9.5 mM (PO 4) <strong>with</strong>out calcium or magnesium,powdered version of 17-512‘15°C–30°C10 l142North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Buffers and Buffered SalinesContinuedBuffered SalineWithoutPhenol RedWithoutCalcium orMagnesiumWith HEPESCat. No.SizePhosphate Buffered Saline17-516F 500 ml ■ ■17-516Q 1 l ■ ■17-517Q 1 l ■ ■UltraSaline A12-747F 500 ml ■ ■For exact compositions, see reagents formulation section, page 439–455.15°C–30°C unless noted otherwise in ordering informationOrdering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size17-737F 17-737F HEPES Buffer (1 M) Contains 238.3 g/L in normal saline 15°C–30°C 500 ml17-737E BE17-737E HEPES Buffer (1 M) Contains 238.3 g/L in normal saline 15°C–30°C 100 mlBE02-018T Phosphatase Buffered Saline ET Used in animal embryo transfer procedures, bottle <strong>with</strong> septum cap 15°C–30°C 1 l17-517Q BE17-517Q Phosphate Buffered Saline (10X) 67 mM (PO 4) <strong>with</strong>out calcium or magnesium (phenol red?) 15°C–30°C 1 l17-516F BE17-516F Phosphate Buffered Saline (1X) 6.7 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 500 ml17-516Q BE17-516Q Phosphate Buffered Saline (1X) 6.7 mM (PO 4) <strong>with</strong>out calcium or magnesium 15°C–30°C 1 lBE02-017F BE02-017F Phosphate Buffered Saline EDTA Used in procedures of generating human dendritic cells from15°C–30°C500 mlmonocytes, pH 7.5 solution04-616F 04-616F TL HEPES Solution 2°C–8°C 500 ml12-747F 12-747F UltraSaline A HEPES buffered saline <strong>with</strong>out phenol red, enhances trypsin action 2°C–8°C500 mlwhen used to rinse monolayer before subculture12-624E 12-624E Veronal Buffer (5X) 15°C–30°C 100 ml17-711E BE17-711E Versene® (EDTA), 0.02% 0.2 g/l Ethylenediaminetetraacetic acid (0.53 mM) in DPBS, <strong>with</strong>outcalcium or magnesium15°C–30°C100 ml2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com143
Viral Serology2Media, Reagents and Sera / BioWhittaker Cell Culture ReagentsComplement fixation (CF) is an immunological test that canbe used to detect the presence of either a specific antibodyor a specific antigen. Complement fixation reagents includeGuinea Pig Complement, Sheep Erythrocytes, andHemolysin. The CF test involves two basic principles:––Complement is irreversibly bound (fixed) to antigenantibodycomplexes. The degree of fixation is governedby the relative concentration of either antigen orantibody.––The lysis of sheep red blood cells in the presence ofhomologous antibody (hemolysin) is dependent uponthe presence of complement.The complement fixation test is interpreted as follows:antigen + serum + complement + sensitized sheep redblood cells––antibody present = no hemolysis––antibody absent = hemolysisInfluenza virus was shown to agglutinate chicken red bloodcells (RBC). Subsequently, a variety of viruses have alsobeen shown to agglutinate RBC’s from several differentspecies. Viruses have been shown to agglutinate sheep redblood cells, chicken red blood cells, and guinea pig red bloodcells in the hemagglutination (HA) assay. It has also beenobserved that specific antibodies can inhibitThe complement fixation test uses sheep red blood cells (sRBC),pre-bound by anti-sRBC antibody, hemolysin, and serum (usually from aguinea pig) as a source of complement. Complement is a system of serumproteins which interact <strong>with</strong> the antigen-antibody complex. This reactioncauses pores to form in the membrane of the cell which ultimately resultsin lysis of the red blood cell.hemagglutination which led to the development of thehemagglutination inhibition (HAI) assay. The HA-HAIcapability provides a fast and easy method of quantifyingboth viral antigen and antibody. The specificity andsensitivity of the HAI assay is dependent upon thecharacteristics of the HA antigen and its interaction <strong>with</strong>antibody, which will vary <strong>with</strong> the particular virus undertest.Ordering Information – BioWhittaker Cell Culture ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size55-402J 55-402J Hemolysin Rabbit anti-sheep erythrocyte serum 100 ml30-956J 30-956J Complement – Guinea Pig Supplied lyophilized <strong>with</strong> restoring solution 5 ml30-904J 30-904J Chicken Red Blood Cells 1 part whole blood, 5 parts Alsever‘s solution 5 ml30-957J 30-957J Guinea Pig Red Blood Cells 1 part whole blood, 5 parts Alsever‘s solution 5 ml55-401A 55-401A Sheep Erythrocytes 40% whole blood, 60% Alsever‘s solution 100 ml144North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
BioWhittaker Animal and Human Sera2Penicillin-Streptomycin Mixtures 141Buffers and Buffered Salines 142Viral Serology 144BioWhittaker Animal and Human SeraIntroduction146Fetal Bovine Sera 149Bovine Sera 149Human Sera 150Fetal Bovine Serum 150Equine Sera 151Media, Reagents and Sera / BioWhittaker Animal and Human SeraEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com145
Introduction2Media, Reagents and Sera / BioWhittaker Animal and Human SeraBioWhittaker Sera and Media Products have been theleaders in quality for over 50 years. Our strict adherence tocurrent Good Manufacturing Practices (cGMP) results in thehighest quality serum products available. From raw materialcollection to final product release, in-process qualityassurance tests are designed to ensure that the productswe supply meet our own high quality standards as well asyour stringent requirements.All US-origin animal sera are collected from abattoirsinpected by the USDA and located <strong>with</strong>in the contiguous 48states. USDA approved source fetal bovine serum(Cat. No. 14-502F) is USDA certified and approved forimportation into the United States.All sera, including bovine, equine and human, are asepticallycollected and must meet established component specificationsbefore they are accepted and released by QualityAssurance for use in further manufacturing. Daily collectionrecords indicate the day, month and year the raw blood wascollected and provide traceability to the individual pool,which provides traceability back to the USDA establishmentnumber and abattoir or donor herd from which the rawmaterial originated.Prior to filtration, the serum is dispensed into a batch tankand mixed to assure bottle-to-bottle homogeneity. All fetalbovine serum is sterile filtered through three 0.1-micronfilters.Quality AssuranceWe produce a variety of serum products for use in cellculture media, production of biological and diagnosticproducts and other laboratory procedures that demand thefinest quality serum available. In order to maintainconsistent quality in serum products, strict qualityassurance of each production lot is maintained.Raw Sera Quality ControlAll individual pools of raw serum are evaluated and mustmeet established specifications before they are releasedfor further manufacturing. Each pool must be free ofdetectable mycoplasma and meet stringent specificationsfor endotoxin, hemoglobin, pH, osmolality and protein. Fetalbovine serum lots are also tested for adventitious bovineviruses prior to acceptance. Only those pools meeting ourstandards of quality are accepted for use in furthermanufacturing.Finished Product Quality ControlSamples of each lot of finished product are selectedfollowing 9CFR guidelines and tested in accordance <strong>with</strong>written final product specifications. Final product qualitycontrol tests include the following:Sterility – Tests are performed on representative samplesusing a modification of the membrane filtration methoddescribed in US Pharmacopeia (USP). A representativesample of serum is filtered. Filters are subsequentlyimmersed in fluid thioglycolate medium (FTM) andtrypticase soy broth (TSB). TSB cultures are incubated at22.5°C ± 2.5°C. FTM cultures are incubated at 32.5°C ±2.5°C. The cultures are incubated for 14 days, during whichthey are periodically examined to ensure absence ofmicrobial contamination.Mycoplasma – Final products are tested for mycoplasmaby two separate methods: the large volume biological test(Barile and Kern) and a DNA staining technique (Hoechstmethod).Adventitious viruses – All bovine serum lots receive fullcompliance 9CFR testing for adventitious viral agentsincluding bovine viral diarrhea (BVD), infectious bovinerhino tracheites (IBR), parainfluenza virus type 3 (PI3),bovine parvovirus, bovine adenovirus 3 and 5, rabies virus,blue tongue, BRSV and reovirus. All donor horse serum lotsreceive full compliance 9CFR testing for adventitious viralagents including BVD, ERV/EHV-1, EVA, EIA, rabies virus,RED, IBR (CPE) and PI3 (hemadsorption).Cell growth promotion – All serum lots of animal origin aretested for their growth promoting properties using primary,diploid, and heteroploid cell lines unless other cells areappropriate based on the product’s use. Results are obtainedusing the Lowry protein method to assess cell replication.Visual screening of cultures is also done to ensurecharacteristic cell morphology and lack of toxicity.146North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionContinuedChemistry – pH, osmolality, total protein and hemoglobindeterminations are performed for each lot of serum.Osmolality is determined by means of the highly repeatablefreezing point method. Total protein is determined by theBiuret method. An extensive panel of chemical tests,including a biochemical profile, is run on each lot of animalserum. The biochemical profile includes:Albumin Gamma Globulin SGOTAlkaline Phosphatase Glucose SGPTBlood Urea Nitrogen Glutamyl Transpeptidase SodiumCalcium % Gamma Globulin Total BilirubinChloride Iron Total ProteinCholesterol Lactic Dehydrogenase TriglyceridesCreatinine Phosphorus Uric AcidFree Fatty Acids PotassiumSerum identity – Samples are tested for characteristicelectro phoretic separation of proteins that appear as visiblebands or precipitate when the appropriate antiserum isallowed to react <strong>with</strong> them. This technique allows verificationof serum specification.Endotoxin – Serum products are routinely tested for thepresence of endotoxin. Testing is done by the QuantitativeChromogenic LAL method (Kinetic-QCL®) to yield a specificvalue reported in endo toxin units (EU/ml). Results of eachlot are available on the Certificate of Analysis issuedfor each lot.Immunoglobulin G – Fetal bovine serum is quantitativelytested by radial immunodiffusion.Bacteriophage – Fetal bovine serum lots are tested for thepresence of bacteriophage.Origin and Types of SeraFetal Bovine Serum, Premium, US Origin – All whole fetalbovine serum is aseptically collected using a cardiacpuncture technique from USDA inspected abattoirs located<strong>with</strong>in the 48 contiguous United States. Each lot iscompletely traceable back to the USDA establishmentnumber from which the raw material was obtained. Donoranimals receive ante – and post-mortem inspection by anon-site USDA veterinary inspector and appear to be free ofinfectious diseases.Fetal Bovine Serum, USDA Approved Source – All whole fetalbovine serum is aseptically collected using a cardiacpuncture technique from USDA approved sources. Each lotproduced is safety tested for exotic viruses and approvedfor importation into the United States by the USDA. Donoranimals receive ante – and post-mortem inspection by anon-site USDA veterinary inspector and appear to be free ofinfectious diseases.Fetal Bovine Serum, Premium, US Origin, Irradiated –Irradiated fetal bovine serum is produced in the samemanner and from the same high quality US origin fetalbovine serum as our premium FBS and irradiated to reduceor destroy any bovine viruses. In addition, the premium FBSis irradiated in its final product container, instead of in anunfinished state to assure that the effectiveness of theirradiation process is not compromised due to furtherproduct packaging after irradiation. Each lot must passcomplete growth promotion testing following irradiation toassure you that the irradiation treatment has notcompromised the growth promoting abilities of serum.Human Serum – US Collection – Whole human blood isobtained from normal human donors who test negative forHepatitis B surface antigen (HBsAg), antibodies to HepatitisC (HCV), and Hum an Immunodeficiency Virus 1 and 2(HIV-1 and HIV-2). Serum is obtained off the clot, centrifugedto remove red blood cells, pooled and sterile filtered.Calf Serum, (Bovine) – Whole bovine calf blood is of USorigin and is collected from animals that are 10 days to 6months of age. Careful handling and quick processingensure that low levels of endotoxin are maintained.Calf Serum, Newborn (Bovine) – Whole newborn calf bloodis of US origin and is collected from animals that are 0–10days of age. Newborn calf serum is a popular alternative tofetal bovine serum.Horse Serum, Donor – US Origin – Whole donor horse bloodis collected from a donor herd located in the United States.Donors are fasted prior to bleeding to reduce lipidconcentration. Each animal receives routine Coggins testsfor equine infectious anemia (EIA).Human Serum, AB – US Collection – Whole human AB bloodis obtained from normal human donors who test negativefor Hepatitis B surface antigen (HBsAg), antibodies toHepatitis C (HCV), and Human Immuno deficiency Virus 1and 2 (HIV-1 and HIV-2). Serum is obtained off the clot,centrifuged to remove red blood cells, pooled and sterilefiltered.Human Serum, AB (Male only) – US Collection – Wholehuman AB blood is obtained from normal human male2Media, Reagents and Sera / BioWhittaker Animal and Human SeraEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com147
IntroductionContinued2Media, Reagents and Sera / BioWhittaker Animal and Human Seradonors who test negative for Hepatitis B surface antigen(HBsAg), antibodies to Hepatitis C (HCV), and HumanImmuno deficiency Virus 1 and 2 (HIV-1 and HIV-2). Serum isobtained off the clot, centrifuged to remove red blood cells,pooled and sterile filtered.Human Serum, AB (Male only), Plasma Derived –US Collection – Human AB serum, derived from plasma, isobtained from normal human male donors who testnegative for Hepatitis B surface antigen (HBsAg), antibodiesto Hepatitis C (HCV), and Human Immunodeficiency Virus 1and 2 (HIV-1 and HIV-2). Serum is pooled and sterile filtered.Handling Serum ProductsAnimal serum varies from lot-to-lot. To minimize thevariability, it must be handled consistently.Receipt – All serum is stored frozen and shipped <strong>with</strong> dryice. A fee will be added to your invoice for dry ice packing. Itis critical that all boxes containing serum be emptied uponreceipt.Storage – We recommend that serum be stored at itslabeled temperature.Using serum – Although the product has been sterilefiltered, aseptic procedures must be followed at all times.Whenever possible use a biological safety cabinet that hasbeen certified according to the National SanitationFoundation Standard 49.Granules, flocculent material or turbidity may develop afterthawing. This particular matter does not alter theperformance of the serum as a supplement for cell culturemedium. Repeated freezing/thawing of serum may increasethe amount of precipitate and is therefore not recommended.If you do not intend to use an entire bottle of serum <strong>with</strong>in2 freeze/thaw cycles, aliquot it into usable quantities insterile containers before freezing a second time.Wipe the outside of each bottle <strong>with</strong> a disinfectant solution,proven effective against the normal bioburden in yourlaboratory, before setting it on the work surface. Removethe heat seal and wipe the outside of the cap <strong>with</strong> thedisinfectant solution.Inactivation of Viruses and Mycoplasma by GammaIrradiationAs commercialization of biological products continues toescalate, regulatory agencies are looking more closely atingredients of animal origin being used in the manufacture ofbiological products.To satisfy the regulatory concerns youface, we have performed a study on the efficiency of usinggamma irradiation to remove bovine viruses from serum.Our process of gamma irradiation delivers a precise dose ofgamma irradiation to each bottle by multi-dimensional dosemapping and standardized handling and packagingconfigurations.Our validation study includes demonstrating up to a six-logreduction in bovine viruses, as well as confirming the serum’sability to support cell growth after irradiation. The completevalidation study is available to review during an on-site auditof Lonza Walkersville, Inc.FBS, US Origin, Irradiated (14-471F) is routinely tested forthe presence of bovine viruses by full compliance 9CFR testprocedures. These results include test results for thefollowing bovine viruses: Bovine Viral Diarrhea (BVD), IBR(CPE), PI3 (hemadsorption), Bovine parvovirus, Bovineadenovirus 3 and 5, Rabies virus, Blue tongue, Bovinesyncitial respiratory virus (BRSV) and Reovirus.In addition, each lot receives our full cell growth testfollowing irradiation and a Certificate of Irradiation isprovided confirming the product received the proper doserequired to inactivate bovine viruses.Special Serum TreatmentsWe will heat inactivate or gamma irradiate serum on anindividual basis. You may designate approved lots to beheat inactivated or gamma irradiated by informing ourCustomer Service when placing an order. There will be anadditional charge for these services. Please allow 4 weeks ormore, for these procedures.Heat Inactivation of SerumUnless there is a compelling reason to heat inactivate yourFBS, we recommend that it is not be done. Heat inactivationwas historically done for many reasons that are no longervalid. Generally, FBS performs better in cell culture <strong>with</strong>outheat treatment.148North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionContinuedWe recommend the following protocol for heatinactivation:1. Place the serum at room temperature for 3–4 hoursbefore thawing the serum completely in a 37°C waterbath.2. Preheat a water bath to 56°C.3. Agitate the bottles to mix the contents. Set them intothe water bath so that the water level is above serumlevel.4. Place a thermometer in a bottle <strong>with</strong> an equivalentvolume of water in the water bath as a control.Fetal Bovine Sera10°C–-20°CBovine Sera10°C–-20°C5. Mix the serum occasionally as the temperature rises.Monitor the temperature of the water bath to ensurethat it stays at 56°C. When the temperature of the waterin the control bottle reaches 56°C, set a timer for30 minutes.6. At regular intervals, ensure that the temperature is56°C and swirl the bottles.7. At the end of 30 minutes, remove the bottles from thewater bath and label them as “heat inactivated”.Ordering Information – Fetal Bovine SerumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size14-501F 14-501F Fetal Bovine Serum – Premium, US Origin Screened for viruses by full compliance 9CFR (113.53)and the absence of mycoplasma, 3 × 0.1-micron filtered14-502F 14-502F Fetal Bovine Serum, USDA Approved Source Screened for viruses by full compliance 9CFR (113.53)and the absence of mycoplasma, 3 × 0.1-micron filtered14-503F 14-503F Fetal Bovine Serum – Premium, US Origin,Heat Inactivated14-507F 14-507F Fetal Bovine Serum – Premium, USDAApproved Source, Heat Inactivated14-471F 14-471F Fetal Bovine Serum – Premium, US Origin,IrradiatedFetal Bovine Serum – Premium,US Origin, IrradiatedFetal Bovine Serum –Premium, US Origin, IrradiatedHeat inactivated at 56°C for 30 minutes, screenedfor viruses by full compliance 9CFR (113.53) and theabsence of mycoplasma, 3 × 0.1-micron filteredHeat inactivated at 56°C for 30 minutes, screenedfor viruses by full compliance 9CFR (113.53) and theabsence of mycoplasma, 3 × 0.1-micron filteredIrradiation dose is 25–40 kGy, screened for viruses(IBR, P13, BVD) and the absence of mycoplasma,3 × 0.1-micron filtered-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°COrdering Information – Bovine SeraCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size14-416E 14-416E Calf Serum - Newborn, US Origin Screened for viruses by full compliance 9CFR and theabsence of mycoplasma, 3 x 0.2-micron filtered-10°C to -20°C500 ml500 ml500 ml500 ml500 ml100 ml2Media, Reagents and Sera / BioWhittaker Animal and Human SeraEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com149
Human Sera10°C–-20°C2Ordering Information – Human SerumCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size14-490E 14-490E Human Serum, AB -10°C to -20°C 100 ml14-498E 14-498E Human Serum, AB Male only -10°C to -20°C 100 ml14-491E 14-491E Human Serum, AB Male only, plasma-derived -10°C to -20°C 100 ml14-490EH 14-490EH Human Serum, AB Heat inactivated -10°C to -20°C 100 mlMedia, Reagents and Sera / BioWhittaker Animal and Human SeraFetal Bovine Serum (Available Outside USA and Canada)The following sera are ONLY available for customers outside the USA and CanadaAll sera listed follow EC regulations on import for serum intothe European community. Products manufactured usingthose sera might be subject to import restrictions in somecountries (e.g., USA).The EC approved fetal bovine sera are manufactured ina European ISO 9001 certified facility.Finished Product Quality ControlSamples of each lot of finished product are selected andtested in accordance <strong>with</strong> written product specifications.Final product quality control includes the following:––Sterility – Tests are performed on representativesamples using a modification of the membrane filtrationmethod described in the EP Pharmacopoeia––Mycoplasma – Final products are tested for mycoplasmausing the culture test described EuropeanPharmacopoeia (EP)––Adventitious viruses – All fetal bovine serum lotsreceive testing for adventitious viral agents includingbovine viral diarrhea (BVD), infectious bovinerhinotracheitis (IBR) and parainfluenza virus type 3(PI3)––Cell growth promotion – All fetal bovine serum lots aretested for their growth promoting properties■■Applications––Support growth of undifferentiated embryonic stemcells––Low unspecific stimulation-10°C–-20°COrdering Information – Fetal Bovine Serum – EU StandardCat. No. EU Product Name Product Description Storage Conditions SizeDE14-801E Fetal Bovine Serum – EU Standard Brazilian Origin, screened for viruses (IBR, P13, BVD) and the absenceof mycoplasmaDE14-801F Fetal Bovine Serum – EU Standard Brazilian Origin, screened for viruses (IBR, P13, BVD) and the absenceof mycoplasmaDE14-701E Fetal Bovine Serum – USDA Country of Origin Australian origin, screened for viruses (IBR, P13, BVD) and theabsence of mycoplasmaDE14-701F Fetal Bovine Serum – USDA Country of Origin Australian origin, screened for viruses (IBR, P13, BVD) and theabsence of mycoplasma-10°C to -20°C-10°C to -20°C-10°C to -20°C-10°C to -20°C100 ml500 ml100 ml500 ml150North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Fetal Bovine Serum (Available Outside USA and Canada)ContinuedOrdering Information – Fetal Bovine Serum – EU Standard, DialyzedCat. No. EU Product Name Product Description Storage Conditions SizeDE14-810E Fetal Bovine Serum – EU Standard, Dialyzed Brazilian Origin, screened for viruses (IBR, P13, BVD) and the absenceof mycoplasma. Dialyzed against physiological NaCl solution or PBSusing a 10 kDa molecular weight cut-off membrane until the glucoselevel is < 10 mg/dl. To prevent precipitation and inactivation of serumpeptides we do not perform exhaustive dialysis.Thus we do not certify that all low molecular weight dialyzablecomponents, such as amino acids, are totally removed by ourprocessing. The dialyzed serum is sterile filtered..DE14-810F Fetal Bovine Serum – EU Standard, Dialyzed Brazilian Origin, screened for viruses (IBR, P13, BVD) and the absenceof mycoplasma. Dialyzed against physiological NaCl solution or PBSusing a 10 kDa molecular weight cut-off membrane until the glucoselevel is < 10 mg/dl. To prevent precipitation and inactivation of serumpeptides we do not perform exhaustive dialysis.Thus we do not certify that all low molecular weight dialyzablecomponents, such as amino acids, are totallyremoved by our processing. The dialyzed serum is sterile filtered..DE14-820EDE14-820FDE14-830FDE14-840EDE14-840FDE14-850FDE14-860FFetal Bovine Serum – EU Standard, CharcoalDextran TreatedFetal Bovine Serum – EU Standard, CharcoalDextran TreatedFetal Bovine Serum – EU Standard,Tetracycline-freeFetal Bovine Serum – EU Standard,DelipidizedFetal Bovine Serum – EU Standard,DelipidizedFetal Bovine Serum – EU Standard, ESQualified SerumFetal Bovine Serum – EU Standard, UltraLowIgG (
Notes2Media, Reagents and Sera152North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>3 Mycoplasma Detection and Prevention3Detection156Elimination and Prevention 159
Mycoplasma Detection and PreventionIntroduction155DetectionMycoAlert PLUS Mycoplasma Detection Kit –For <strong>Research</strong> Use 157MycoTOOL® PCR Mycoplasma Detection Kit –For Final Release Testing 1583Elimination and PreventionMycoZap Mycoplasma Elimination Reagent 160MycoZap Antibiotics 161MycoZap Spray 162Mycoplasma Detection and Prevention154North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionOne of the most common contaminants present in cellculture laboratories are mycoplasma. A conservativeestimate states that between 15–35% of all continuous cellcultures are contaminated <strong>with</strong> mycoplasma 1 , someestimates are even higher (up to 80 % in some countries) 2 .What are Mycoplasma?––Belong to the family Mollicutes including Mycoplasma,Acholeplasma, Ureaplasma, Entomoplasma,Spiroplasma and other species––Smallest free-living, self-replicating organisms (size:0.2 μm–0.8 μm)––Simple prokaryocytes, lacking a rigid cell wall(surrounded by a single plasma membrane)––Usually attached to the external surface of the cellmembrane––Relying on their hosts for many nutrients as theirbiosynthetic capabilities are limited––Over 180 recognized species––Six species account for 95% of all mycoplasmainfections in cell cultures (M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii)––Widespread in nature as parasites of humans,mammals, reptiles, insects and plantsTypical Routes of Mycoplasma Infection in Cell Cultures––Cross contamination from untested infected cells––Aerosols created during pipetting––Using the same bottle of medium for different cell types––Handling more than 1 cell line in the hood at a time––Contaminated materials––Contaminated donor tissue (10 7 cfu/ml––Most routine antibiotics used in cell culture areineffective against mycoplasma––They are not routinely removed by filtrationwww.lonza.com/mycoplasmaHow Do Mycoplasma Affect Your Cells?Increased sensitivity toinducers of apoptosisChromosomalaberrationsDisruption of nucleicacid synthesisChanges in cellmembrane antigenicityMycoplasma contamination can seriously impact thereliability, reproducibility, and consistency of experimentalresults, representing a major problem for basic research aswell as for the manufacturing of bioproducts. Standardtesting for mycoplasma is an important quality control thatshould be included in cell culture protocols. We provide apowerful product offering for reliable detection andsuccessful elimination and prevention of mycoplasmacontamination:––MycoAlert Mycoplasma Detection Kit – Accurate,reliable and universal mycoplasma detection––MycoTOOL® PCR Mycoplasma Detection – Rapidmycoplasma testing kits and services for final productrelease testing––MycoZap Mycoplasma Elimination Reagent –Successful elimination of mycoplasma <strong>with</strong> low celltoxicity––MycoZap Spray – Reliable disinfection of laboratorysurfaces from mycoplasma contamination (available inEurope only)––MycoZap Prophylactic – Prevention of mycoplasmacontamination in combination <strong>with</strong> your antibioticformula of choice––MycoZap Plus-CL and Plus-PR – Protection against abroad range of microbial contaminants, such asGram(+) and Gram(-) bacteria, fungi and mycoplasmaReferencesCell deathInhibition of cell growthAlteration of DNAtransfection efficiencyInhibition of cellmetabolismCompromisedproduction of virusesDNA fragmentation due tomycoplasma nucleases, NOTapoptosis1. Drexler H.G., Uphof C.C. (2002): Mycoplasma contamination of cellcultures: Incidence, sources, effects, detection, elimination,prevention. Cytotechnology 39: 75–90.2. Koshimizu K., Kotani H. (1981) in: Procedures for the Isolation andIdentification of Human, Animal and Plant Mycoplasmas (NakamuraM., ed.), Saikon, Tokyo, 87-102.3Mycoplasma Detection and PreventionEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com155
Detection3Mycoplasma Detection and Prevention / DetectionDetectionMycoAlert PLUS Mycoplasma Detection Kit –For <strong>Research</strong> Use 157MycoTOOL® PCR Mycoplasma Detection Kit –For Final Release Testing 158156North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
MycoAlert PLUS Mycoplasma Detection Kit – For <strong>Research</strong> UseThe MycoAlert Assay is a selective biochemical test thatexploits the activity of mycoplasmal enzymes which arefound in all six of the main mycoplasma cell culturecontaminants and the vast majority of 180 mycoplasmaspecies, but are not present in eukaryotic cells. Viablemycoplasma in a test sample are lysed and the enzymesreact <strong>with</strong> the MycoAlert PLUS Substrate, catalyzing theconversion of ADP to ATP. By measuring the level of ATP in asample via a luciferase reaction, both before (read A) andafter the addition of the MycoAlert PLUS Substrate (readB), a ratio can be obtained which is indicative of thepresence or absence of mycoplasma. The new nextgeneration MycoAlert PLUS Assay generates a strongerlight signal and thus provides broader compatibility <strong>with</strong>plate luminometers and multifunctional readers.■■Benefits––Results in 1 equals mycoplasma contaminationReading AReading B10 2 4 6 8 10 12 14 16Time (minutes)M hyorhinis infected: B/A ratio 114 Uninfected control: B/A ratio
MycoTOOL® PCR Mycoplasma Detection Kit – For Final Release TestingRapid mycoplasma detection kits and testing servicesusing the MycoTOOL® PCR Mycoplasma Detection Kit fromRoche are now available through Lonza. Accepted by theEuropean Medicines Agengy (EMA) and US Food and DrugAdministration (USFDA), the MycoTOOL® PCR test is the firstcommercially available assay to replace traditional, timeconsumingmycoplasma culture tests for release testing ofseveral pharmaceutical products.3Mycoplasma Detection and Prevention / Detection■■Benefits––Results in hours instead of days––Only regulatory accepted PCR-based method for finalproduct release testing––Reliable assay controls in place to help ensure validityof test result––Predicted to detect more than 90 mollicute species dueto universal primer design, including Mycoplasma,Spiroplasma, and Acholeplasma––Validated down to 1 CFU/ml at a 95% confidence level<strong>with</strong> more than 10 species of mycoplasma■■Applications––Final product release test method for mycoplasmadetection––Suited for both cellular and cell-free matrices: vaccines,media/sera, cell culture supernatantsMycoplasma species validated down to 1 CFU/ml at a95% confidence level (≥23 out of 24 positive)Species Claimed sensitivity Probit analysis ofvalidation data1 CFU/ml CFU/mlA. laidlawii* +
Elimination and Prevention3Elimination and PreventionMycoZap Mycoplasma Elimination Reagent 160MycoZap Antibiotics 161MycoZap Spray 162Mycoplasma Detection and Prevention/ Elimination and PreventionEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com159
MycoZap Mycoplasma Elimination Reagent3Mycoplasma Detection and Prevention/ Elimination and PreventionThe MycoZap Reagent can eliminate detectablemycoplasma contamination in as few as 4 days and hasbeen optimized to clear mycoplasma <strong>with</strong> minimal toxiceffects on the infected cells. It eliminates mycoplasma byusing a combination of antibiotic and antimetabolic agents.This approach allows for a highly reliable elimination ofmycoplasma that cannot be achieved by the use ofantibiotics alone. The MycoZap Reagent can be used toeradicate mollicutes, including Mycoplasma, Acholeplasma,Spiroplasma and Entomoplasma species in cell cultures.■■Benefits––Efficient mollicute elimination by a combination ofantibiotic and antimetabolic agents––Minimal toxic effects on cells■■Applications––Eradicates Mycoplasma, Acholeplasma, Spiroplasma,and Entomoplasma––Suited for a broad range of cell cultures2°C–8°Cwww.lonza.com/mycoplasmaEfficient Mycoplasma Removal <strong>with</strong> Minimal Effect onCell ViabilityMycoAlert ratio100Mycoplasma detected101No mycoplasma detected0.1Day 0 Day 4 Day 8 Day 11 Day 17untreateduntreated viabilitytreatedtreated viability100000001000000100000100001000The MycoZap Reagent treatment eliminates mycoplasma in as few as4 days (detected by MycoAlert Assay) <strong>with</strong> minimal impact on cellviability (determined by ViaLight Assay).Ordering Information – ReagentCat. No. NA Cat. No. EU Product Name SizeLT07-818 LT07-818 MycoZap Mycoplasma Elimination Reagent 1 treatmentLT07-918 LT07-918 MycoZap Mycoplasma Elimination Reagent 5 treatmentsRelated ProductsMycoAlert PLUS Mycoplasma Detection Kit 157MycoZap Antibiotics 161MycoZap Spray 162100101ViaLight Plus RLUsPage160North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
MycoZap AntibioticsMycoZap Antibiotics are extremely powerful combinationsof innovative antibiotics for the protection of cell culturesfrom mycoplasma contamination. While MycoZapProphylactic prevents mycoplasma contamination,MycoZap Plus offers complete protection against a broadrange of common contaminants including mycoplasma.MycoZap Prophylactic■■Benefits––Specifically prevents mycoplasma contamination––Also active against other species of the mycoplasmagroup like Acholeplasma and Spiroplasma3■■Applications––Can be used in combination <strong>with</strong> other antibiotics (e.g.,Pen/Strep) to prevent other microbial contaminantsMycoZap Plus-CL and MycoZap Plus-PR■■Benefits––Active against mycoplasma, Gram(-) and Gram(+)bacteria as well as yeast and fungi––Complete solution replacing Pen/Strep formulation■■Applications––MycoZap Plus-CL for protection of cell lines––MycoZap Plus-PR optimized for gentle protectionof primary cellsImmediate use: 2°C–8°CLong-term storage: below -18°Cwww.lonza.com/mycoplasmaPrevention againstmycoplasmaPrevention against– Gram(+) bacteria– Gram(-) bacteria– Fungi– YeastMycoplasma OnlySolutionMycoZapProphylacticComplete SolutionsMycoZapPlus-CLMycoZapPlus-PR■ ■ ■No; but can be usedin combination <strong>with</strong>other antibioticformula of choiceSuited for primary cells ■ ■Suited for cell lines ■ ■Ordering Information – ReagentCat. No. NA Cat. No. EU Product Name SizeVZA-2011 VZA-2011 MycoZap Plus-CL Antibiotic 10 × 1 mlVZA-2012 VZA-2012 MycoZap Plus-CL Antibiotic 1 × 20 mlVZA-2021 VZA-2021 MycoZap Plus-PR Antibiotic 10 × 1 mlVZA-2022 VZA-2022 MycoZap Plus-PR Antibiotic 1 × 20 mlVZA-2031 VZA-2031 MycoZap Prophylactic 10 × 1 mlVZA-2032 VZA-2032 MycoZap Prophylactic 1 × 20 mlRelated ProductsPageMycoAlert PLUS Mycoplasma Detection Kit 157MycoZap Mycoplasma Elimination Reagent 160MycoZap Spray 162■■Mycoplasma Detection and Prevention/ Elimination and PreventionEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com161
MycoZap Spray (Europe Only)3Mycoplasma Detection and Prevention/ Elimination and PreventionMycoZap Spray is an effective solution for reliabledisinfection of laboratory surfaces and apparatus, such asclean benches, incubators, cell storage boxes and liquidnitrogen containers. MycoZap Spray shows an excellenteffectiveness against mycoplasma. A special biologicalingredient shows an extreme affinity to mycoplasma anddevelops its full activity together <strong>with</strong> the alcoholic anddetergent properties of MycoZap Spray. Clean andcontamination-free surfaces can be achieved in a singleapplication.■■Benefits––Eradicates mycoplasma <strong>with</strong>in seconds––Non-corrosive and non-carcinogenic––Convenient, ready-to-use and stable■■Applications––Effective against a broad range of organisms includingenveloped and non-enveloped viruses, Gram(+) andGram(-) bacteria as well as protozoaRoom temperaturewww.lonza.com/mycoplasmaOrdering Information – ReagentCat. No. EU Product Name Product Description SizeVZA-2001 MycoZap Spray 500 mlVZA-2002 MycoZap Spray, Refill 1 lRelated ProductsPageMycoAlert PLUS Mycoplasma Detection Kit 157MycoZap Antibiotics 161162North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>4 Transfection4Nucleofector Technology 165Nucleofector Devices and Systems 171Nucleofector Kits 177Nucleofector Kit Accessories 239
Transfection4Nucleofector TechnologyIntroduction166Components of the Nucleofector Technology 168Advanced Platform: 4D-Nucleofector System 169Adherent Nucleofection 170Nucleofector Devices and Systems4D-Nucleofector System 17296-well Shuttle System 173Nucleofector 2b Device 174384-well Nucleofector System 175Nucleofector KitsNucleofector Kits for Primary Cells – OverviewPrimary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 178Adherent Nucleofector Kits for4D-Nucleofector System 182Primary Cell Optimization Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 183Primary Cell Kits for Nucleofector II /2b Device 184Nucleofector Kits for Primary AdipocytesNucleofector Kits for Human Pre-Adipocytes 186Nucleofector Kits for Primary Blood CellsNucleofector Kits for Human B Cells 187Nucleofector Kits for Stimulated Mouse B Cells 188Nucleofector Kits for Human Dendritic Cells 189Nucleofector Kits for Mouse Dendritic Cells 190Nucleofector Kits for Human Macrophages 191Nucleofector Kits for Mouse Macrophages 192Nucleofector Kits for Human Monocytes 193Nucleofector Kits for Human Natural Killer Cells 194Nucleofector Kits for Human T Cells 195Nucleofector Kits for Mouse T cells 196Nucleofector Kits for Mammalian Blood Cells 197Nucleofector Kits for Primary Bone /Cartilage CellsNucleofector Kits for Human Chondroycytes 198Nucleofector Kits for Primary Cardiac CellsNucleofector Kits for Rat Cardiomyocytes 199Nucleofector Kits for Primary Dermal CellsNucleofector Kits for Human Keratinocytes 200Nucleofector Kits for Human Melanocytes 201Nucleofector Kits for Primary Endothelial CellsNucleofector Kits for Human Coronary ArteryEndothelial Cells 202Nucleofector Kits for Human MicrovascularEndothelial Cells – Lung 203Nucleofector Kits for Human Umbilical VeinEndothelial Cells 204Nucleofector Kits for Mammalian Endothelial Cells 205Nucleofector Kits for Primary Epithelial CellsNucleofector Kits for Human BronchialEpithelial Cells 206Nucleofector Kits for Human MammaryEpithelial Cells 207Nucleofector Kits for Mammalian Epithelial Cells 208Nucleofector Kits for FibroblastsNucleofector Kits for Human Dermal Fibroblasts 209Nucleofector Kits for Mouse Embryonic Fibroblasts 210Nucleofector Kits for Mammalian Fibroblasts 211Nucleofector Kits for Primary HepatocytesNucleofector Kits for Human Hepatocytes 212Nucleofector Kits for Mouse or Rat Hepatocytes 213Nucleofector Kits for Primary Muscle CellsNucleofector Kits for Human Aortic SmoothMuscle Cells 214Nucleofector Kits for Human SkeletalMuscle Myoblasts 215Nucleofector Kits for Mammalian Smooth Muscle Cells216Nucleofector Kits for Primary Neural CellsNucleofector Kits for Chicken Neurons 217Nucleofector Kits for Mouse Neurons 218Nucleofector Kits for Rat Neurons 219Nucleofector Kits for Mammalian Neurons 220Nucleofector Kits for Mammalian Glial Cells 221Nucleofector Kits for Primary Stem CellsNucleofector Kits for Human CD34 + Cells 222Nucleofector Kits for Human H9 Stem Cells 223Nucleofector Kits for Human MesenchymalStem Cells 224Nucleofector Kits for Human Stem Cells 225Nucleofector Kits for Mouse Embryonic Stem Cells226Nucleofector Kits for Mouse Neural Stem Cells 227Nucleofector Kits for Rat Neural Stem Cells 228Nucleofector Kits for Animal Stem Cells 229Nucleofector Kits for Cell LinesCell Line Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 230Cell Line Optimization Kits for 4D Nucleofector,96-well Shuttle and 384-well Nucleofector Systems 233Cell Line Kits for Nucleofector II /2b Device 234Cell Line Optimization Kit forNucleofector II/2b Device 237Nucleofector Kits for ParasitesBasic Parasite Nucleofector Kits 238Nucleofector Kit AccessoriesIntroduction240Nucleofector PLUS Supplements 241Mouse T Cell Nucleofector Medium 242pmaxCloning Vector 243164North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector TechnologyNucleofector TechnologyIntroduction166Components of the Nucleofector Technology 168Advanced Platform: 4D-Nucleofector System 169Adherent Nucleofection 1704Transfection / Nucleofector TechnologyEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com165
Introduction4Transfection / Nucleofector TechnologyThe application of systems biology and multidisciplinaryapproaches require that cells and model systems displayin vivo like cellular functionality. This means that the futureof cell transfection is in using primary cell types, and thattransfecting these physiologically relevant cell types istypically a very difficult task using traditional methods.Additionally, when using relevant cell lines as modelsystems, the critical issues are to achieve reproduciblyefficient transfection <strong>with</strong> high levels of viability whilematching throughput capability <strong>with</strong> the number oftransfections required at each project phase – from proof ofconcept, through to scale-up and screening-like approaches.With the Nucleofector Technology primary cells and stemcells, as well as cell lines, can be consistently transfected athigh efficiency.Developed in 1998, the Nucleofector Technology wasintroduced to the research market in 2001 as the firstefficient non-viral transfection method for primary cells andhard-to-transfect cell lines. Since then the technology hasevolved through constant innovation.Nucleofector Technology – the Superior Non-viral MethodSSCNucleofectionGFPStandard electroporationNucleofector Technology – the superior non-viral method. Transfection ofthe human natural killer cell line NKL using traditional electroporation andNucleofection. 5 × 10 6 NKL cells were transfected <strong>with</strong> 2.5 μg of pmaxGFPVector. Nucleofection: Nucleofector Solution V; Program O-017. Standardelectroporation: 25 mV, 96 μF. Transfection efficiency was monitored byflow cytometry after 24 hours. Cells transfected by Nucleofection show asignificantly better transfection efficiency compared to cells transfectedby traditional electroporation. Cell viability, as measured 18 hours aftertransfection was also superior using Nucleofection. (Data courtesy ofDr. John Coligan, Laboratory of Immunogenetics, NIH/NIAID, Rockville, MD,USA. J Immunol Methods (2004) 284: 133-140.)SSC30.31% 2.75%GFPThe PrincipleNucleofection is a technology based on the momentarycreation of small pores in cell membranes by applying anelectrical pulse. The comprehensive way in whichNucleofector Programs and cell-type specific solutions aredeveloped enables nucleic acid substrate delivery not onlyto the cytoplasm, but also through the nuclear membraneand into the nucleus. This allows for high transfectionefficiencies up to 99% and makes the transfection successindependent from cell proliferation.DNA Delivery Straight Into the NucleusACDNA delivery straight into the nucleus. Normal human dermal fibroblasts(neonatal) were transfected <strong>with</strong> 2.5 μg R-labeled plasmid DNA encodingeGFP. After 2 hours, cells were fixed <strong>with</strong> 3.5% PFA and analyzed by confocalmicroscopy. R label is shown in (A), GFP fluorescence in (B), DAPI nuclearstaining in (C) and a merge of all 3 fluorescent labels in (D).BD166North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IntroductionContinuedWhat Benefits are Important for Your Work?■■Superior transfection performance?––Electrical parameters are optimized to gain hightransfection efficiency and retain highest viability––Excellent preservation of the physiological status oftransfected cells■■Easy-to-use technology?––More than 650 cell-type specific protocols lead to directtransfection success <strong>with</strong> a multitude of different celltypes––Easy optimization protocols for cell lines and primarycells allow for quick and streamlined optimization ofvirtually any cell type––Dedicated White Papers support numerous applications,such as siRNA transfection and transfection of neurons––Excellent technical and applicative support?■■Highly-skilled Scientific Support Team to assist you inyour research––Scientific Support Team members have a masters orPhD level education in biology, biochemistry orbiotechnology––Many of them <strong>with</strong> over 10 years experience intransfection support■■Proven and innovative technology?––More than 4000 peer-reviewed publications andthousands of systems placed worldwide■■Modularity of the 4D-Nucleofector System allowseasy adaptation to new applications––Invention of Nucleofection of cells in adherence■■Using various cell numbers for different applications?––Nucleofection of 2 × 10 4 to 2 × 10 7 cells are feasible<strong>with</strong>in one single device––Transferability of protocol conditions from small tolarger cell numbers <strong>with</strong> the new 4D-NucleofectorSystem■■Easy expansion of your research?––Explore complex systems by using the same conditionsto deliver DNA, RNA, oligonucleotides, PNA, peptides, orproteins––Different device platforms fulfill your choice of samplethroughput from 1 through 384 transfections per runincluding automated high-throughput■■Avoiding cross-contamination?––Disposable, sterile Nucleofection Vessels minimize therisk of cross-contamination <strong>with</strong> cell or substrateleftoverswww.lonza.com/celldatabasewww.lonza.com/citations% Transfection EfficiencyA1009080706050403020100hES Cell line H9hES Cell line BG01VhES Cell line H9.2hES Cell line HUES 9Human MSCHuman NSCExemplary transfection efficiency data for primary cells and human stemcells.% Positive CellsB1009080706050403020100SSEA4/GFPConserving functionality – the first step to meaningful results. Human H9ES cells preserve pluripotency post Nucleofection. H9 cells weretransfected by Nucleofection <strong>with</strong> the pmaxGFP Vector. (A) Cellsanalyzed after 24 hours show expression of GFP (green) as well as of thepluripotency markers SSEA4 (red) and Oct4 (purple). The blue signalsrefer to nuclear staining by DAPI. (B) The percentage of double-positivecells (GFP/SSEA) was analyzed by flow cytometry. (Data kindly providedby Jennifer Moore, Rutgers University, Piscataway, USA.)System biologyPeptideAntibodiesExpressionProteinProteinConjugatesMouse NSCRat hippocampalneuronsRat NSCPlasmidshRNADrugCandidatesSmallMoleculesFluorescentconjugatesDrug discoveryNucleofector Technology – delivers the widest range of substrates.Overview of substrates that can be transfected into primary cells and celllines using Nucleofection.DNASSEA4Rat cortical neuronsMouse glial cellsPremiRNAAntisenseOligosRNAmRNAHuman T-cells(unstimulated)GFPsiRNAmiRNAHUVECPBMCStructural genomicsHumanPre AdipocytesRat cardiomyocytesFunctionalgenomics4Transfection / Nucleofector TechnologyEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com167
B RhE l f N l f i V llP o u t e c p i nE f N l f i f G 3 llR d i ( ) C l i D N lS d liB RhP o uc D s r t onR d K p y ( ) E C Li D N l f . X iS d b i NComponents of the Nucleofector Technology4The Nucleofector Technology relies on the combination of aNucleofector Device and cell specific Nucleofector Kits:––The Nucleofector Device delivers unique electricalparameters. The electrical settings are pre-programmedfor each optimized cell type and can be selected via thedevice or PC software. We offer three different deviceplatforms plus an add-on device (see table below)––The Nucleofector Kits contain a specific NucleofectorSolution and Supplement, specified cuvettes, pipettes,and the pmaxGFP Control Vector. All NucleofectorSolutions provide a protective environment that allowsfor high transfection efficiency and cell viability, whilehelping to maintain physiologically relevant cellularfunctions. A collection of Nucleofector Kits <strong>with</strong>optimized protocols for primary cells and cell lines isavailableNucleofector KitsNucleofector DeviceHighlyEfficientTransfection4D uc eo ec or P o oco for ero el sFor D Nu l of ct r X Un t ra sf ct on n susp ns onD Nu le fe tor Pro oc l f r MG 63 Ce sor 4D Nuc eo ec or X U it Tr ns ect on n susp ns onOptimized ProtocolsTransfection / Nucleofector Technology––Besides providing optimal Nucleofection Conditions,Optimized Protocols offer comprehensive guidance,including tips for cell sourcing, passage, growthconditions and media, and post transfection cultureOverview of Nucleofection PlatformsAdvanced Platform 96-well Add-on High-throughput Platform Basic DeviceDevice 4D-Nucleofector System 96-well Shuttle Device 384-well Nucleofector Nucleofector 2b DeviceSystemThroughput (samples per run) Low to medium (1-16) Low to high (1-96) High (384) Low (1)Reaction volume 20 µl + 100 µl 20 µl 20 µl 100 µlElectrode material Conductive polymer Conductive polymer Conductive polymer AluminumLow cell numbers (20 µl) 2 × 10 4 to 1 × 10 6 2 × 10 4 to 1 × 10 6 2 × 10 4 to 1 × 10 6 –High cell numbers (100 µl) 2 × 10 5 to 2 × 10 7 – – 2 × 10 5 to 2 × 10 7DNA Vector amount/sample0.2–1 μg (20 µl)0.2–1 μg 0.2–1 μg 1–5 μg1–5 μg (100 µl)siRNA amount/sample0.04–40 pmol (20 µl) 0.04–40 pmol 0.04–40 pmol 0.2–200 pmol(concentration 2 nM – 2 µM)0.2–200 pmol (100 µl)Adherent Nucleofection ■ – – –Compatibility <strong>with</strong> 96-well Shuttle Device ■ – – –168North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Advanced Platform: 4D-Nucleofector SystemBased on user feedback, our engineers and scientists havedeveloped the new innovative 4D-Nucleofector System.This system is designed for maximum flexibility and enablesNucleofection of cells in several formats combined <strong>with</strong>advanced performance and convenience. Due to its modulardesign the 4D-Nucleofector System is extremely flexible inregard to the supported applications.The operation software allows you to design and saveindividual experimental setups. Additionally, a PC editor isavailable that enables predefinition of experiments on a PCwhich can then be uploaded to the 4D-Nucleofector CoreUnit via the integrated USB port.Hardware and Software ComponentsThe 4D-Nucleofector System is a modular systemcomprising of one Core Unit and the X Unit and Y Unit as thefirst available functional units:––Core Unit – Controlling the 4D-Nucleofector System––X Unit – Supporting Nucleofection of various cellnumbers in different formats––Y Unit – Enabling adherent Nucleofection in 24-wellculture platesFor ordering information and further details, pleaserefer to page 172.4D Nucleofector System■■What Benefits are Important for Your Work?––Using different cell numbers for different applications?––Same protocol for 100 μl and 20 μl transfection volume––100 μl Nucleocuvette for high cell numbers up to2 × 10 7––20 μl Nucleocuvette Strip for low cell numbers down to2 × 10 4■■Working <strong>with</strong> various throughputs?––Flexible throughput from 1 to 16 samples––Parallel processing of one or two 100 μl Nucleocuvettes––Pre-programming of settings for up to 50 single 100 μlNucleocuvettes or one 20 μl Nucleocuvette Strip––Kit costs tailored to your throughput■■Transfecting different primary cell types?––Five primary cell kits covering a broad range of primarycells––New Primary Cell Optimization Kit for cells lacking anoptimized protocol––Easy optimization of a variety of cell lines using the96-well Shuttle Add-on System■■Preserving cell functionality?––Adherent Nucleofection of neurons at laterdevelopmental stages––No release of metal ions due to conductive polymerelectrodes4Transfection / Nucleofector TechnologyEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com169
Adherent NucleofectionElectroporation-based methods have so far required cells tobe in suspension for transfection. The NucleofectorTechnology entered a new era and allows directNucleofection of cells in adherence. Cells which typicallygrow in adherence in cell culture, can be kept andtransfected by Nucleofection in their physiological state.The Y Unit of the 4D-Nucleofector System works <strong>with</strong>disposable conductive polymer Dipping Electrode Arraysthat can be inserted into standard 24-well culture plates forNucleofection.4Transfection / Nucleofector Technology■■Benefits––Pre- and post Nucleofection culture in 24-well cultureplates––Nucleofection of cells at any time point during thisculture period, i.e. at a later developmental stage––Transfection efficiencies up to 70% combined <strong>with</strong> highviabilities––Compatible <strong>with</strong> Clonetics Primary Animal Neurons■■Applications––Enables Nucleofection of cells in adherence in 24-wellculture platesFor ordering information and further details, pleaserefer to pages 172 and 182.www.lonza.com/adherent-nucleofectionEfficient adherent Nucleofection of neurons in 24-well culture plates.Mouse cortical neurons were seeded into poly-D-lysine coated 24-wellplates (1 × 10 5 cells/well). After 6 DIV, cells were transfected <strong>with</strong>pmaxGFP Vector using the AD1 4D-Nucleofector Y Kit. One day postNucleofection, cells were stained by MAP2 antibody (red) and analyzedby fluorescence microscopy for maxGFP protein expression.Efficient adherent Nucleofection of endothelial cells in 24-well cultureplates. Human umbilical vein endothelial cells (HUVEC) were isolated andplated in passage 1 into collagen-coated 24-well plates at a density of50,000 cells/well. After 1DIV cells were transfected <strong>with</strong> 16 μg pmaxGFPVector using AD1 4D-Nucleofector Y Solution and program CA-215. Cellswere analyzed for maxGFP Protein expression after 24h. (Data kindlyprovided by M. Sauvage, Pharmaceutical Industry, FR)170North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Devices and SystemsNucleofector Devices and Systems4D-Nucleofector System 17296-well Shuttle System 173Nucleofector 2b Device 174384-well Nucleofector System 175Nucleofector Kits for Human Chondroycytes 1984Transfection / Nucleofector DevicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com171
4D-Nucleofector SystemThe 4D-Nucleofector System is a modular systemcomprising one Core Unit and the X Unit and/or Y Unit as thefirst available functional units, each suited for differentapplications.The Core Unit■■Benefits––Controls up to five functional units––5.7’’ foldable touch screen to operate the system––Intuitive operation software for designing and savingindividual experimental setups––USB port for software update and data transfer––Comprises USB and serial connectivity for the 96-wellShuttle Add-on4Transfection / Nucleofector Devices■■Applications––Controlling the 4D-Nucleofector SystemThe X Unit■■Benefits––Features positions for 20 μl Nucleocuvette Strips and100 μl single Nucleocuvette––Seamless transfer of conditions between differentNucleofection Vessels––Comprises HV connectivity for the 96-well ShuttleSystem––Electrically driven drawer for cuvette retainer■■Applications––Supporting Nucleofection of various cell numbers indifferent formatsTechnical SpecificationsDimensions (w × d × h)45 × 28 × 10.5 cm (17.7 × 11.0 × 4.1 in)System comprising Core Unit and one functionalUnit assembled side-by-sideWeight8.0 kg (17.8 lb)System comprising Core Unit and one functionalUnitPower supply100–110 VAC or 230 VAC, 50–60 Hz,self-regulatingPower consumption 140 VAProtection IP 20The Y Unit■■Benefits––Features positions for one 24-well culture plate <strong>with</strong>inserted Dipping Electrode Array––Electrically driven drawer for plate retainer■■Applications––Enables Nucleofection of cells in adherence in 24-wellculture plateswww.lonza.com/4d-nucleofectorOrdering Information – DevicesCat. No. NA Cat. No. EU Product Name Product DescriptionAAF-1001B AAF-1001B 4D-Nucleofector Core UnitAAF-1001X AAF-1001X 4D-Nucleofector X Unit Requires the Core Unit to build complete systemAAF-1001Y AAF-1001Y 4D-Nucleofector Y Unit Requires the Core Unit to build complete systemAWC-1001 AWC-1001 4D-Nucleofector Service Contract Valid for 1 year, can be purchased at any time during the guarantee period.Comprises diagnosis and repair of the device including all replacement parts andcosts for return shipments of repaired device.AWE-1002 AWE-1002 4D-Nucleofector Guarantee Extension Valid for 2 years, has to be purchased together <strong>with</strong> the system172North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
96-well Shuttle SystemThe 96-well Shuttle System is a medium-throughput addon for the 4D-Nucleofector System suited for convenientoptimization of Nucleofection Conditions or as an assayestablishment tool. The complete system consists of threecomponents:––The 4D-Nucleofector System (Core Unit and X Unit)serving as the program delivery unit––The 96-well Shuttle System which mediates thetransfer of the respective 96-well program to a specificwell of the 96-well Nucleocuvette Plate––A laptop computer <strong>with</strong> the 96-well Shuttle Softwarecontrolling the interaction between the devices■■Benefits––Up to 96 independent programs can be run per plate,processed automatically in
Nucleofector 2b DeviceThe Nucleofector Device is the single cuvette based systemthat has been used in research labs since 2001. It allowsefficient transfection of hard-to-transfect cell lines andprimary cells <strong>with</strong> different substrates (e.g., DNA vectors orsiRNA oligonucleotides) in low-throughput format. TheNucleofector II/2b Device can also be used for bacteriatransformation by using alternative cuvettes.4Transfection / Nucleofector Devices■■Benefits––Highly efficient transfection of primary cells and celllines––Reliable results due to high viability and preservation ofcell functionality––Over 150 ready-to-use Optimized Protocols containingcell-type specific guidance■■Applications––Low-throughput transfection in single cuvette format––Transfection of plasmid DNA, siRNA, shRNA, miRNA, RNAand more, e.g., Morpholinos––Transfection of peptides, proteins or small molecules––Approaching 4,000 peer-reviewed publications––Suited for bacteria transformationTechnical SpecificationsDimensions (w × d × h) 30 × 23 × 11 cm (11.81 × 9.06 × 4.33 in)Weight2.8 kg (6.2 lb)Power supply100–110 VAC or 230 VAC50–60 Hz, self-regulatingPower consumption 50 VA/fuse T630mA L250VProtection IP 20, EN 61010-1, UL 61010A-1www.lonza.com/protocolsOrdering Information – DevicesCat. No. NA Cat. No. EU Product Name Product Description SizeAAB-1001 AAB-1001 Nucleofector 2b DeviceAWD-2002 AWD-2002 Nucleofector 2b Guarantee Extension Valid for 2 years, has to be purchased together <strong>with</strong>the systemAWA-2001 AWA-2001 Nucleofector 2b Service Contract Valid for 1 year, can be purchased at any time duringthe guarantee period. Comprises diagnosis andrepair of the device including all replacement partsand costs for return shipments of repaired device.VKA-1001 VKA-1001 Electroporation Cuvettes for Bacteria (1 mm gap) 50 cuvettes174North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
384-well Nucleofector SystemThe new 384-well Nucleofector System is an independentplatform for high-throughput Nucleofection in a 384-wellformat. With an extremely fast plate processing time of 1minute per plate, it is perfectly suited for screeningapplications <strong>with</strong> maximum reproducibility.The 384-well Nucleofector System consists of a PowerSupply Unit generating the high voltage pulses, a PlateHandler Unit and an intuitive PC-based Operation Software.The 384-well Nucleofector Kits use existing 96-wellShuttle Protocols <strong>with</strong> newly developed conductivepolymer 384-well Nucleocuvette Plates. For an automatedNucleofection Process that requires long-term storage ofcells in Nucleofector Solution, some cells may requirespecialized Automation Kits.■■Benefits––Processes a 384-well plate in 1 minute––Uses existing 96-well Shuttle Protocols––Intuitive PC-based Operation Software■■Applications––High-throughput Nucleofection of low cell numbersdown to 2 × 10 4 cells––Seamless integration into automated liquid handlingenvironmentsContact Scientific Support for exact guidance regardingoptimal kit use.Power Supply Unit (left), and Plate Handler Unit (right), <strong>with</strong> loaded384-well Nucleocuvette PlateTechnical SpecificationsDimensions (w × d × h)WeightOrdering Information – SystemCat. No. NA Cat. No. EU Product Name Product Description384-well Nucleofector Plate Handler:40 cm × 42 cm × 15 cm (15.7 × 16.5 × 5.9 in)384-well Nucleofector Power Supply:13.5 cm × 50 cm × 45 cm (5.3 × 19.6 × 17.7 in)384-well Nucleofector Plate Handler:10 kg (22.04 lb)384-well Nucleofector Power Supply:14 kg (30.86 lb)AAU-1001 AAU-1001 384-well Nucleofector System Includes power supply, plate handler, laptop, and softwareAWU-1001 AWU-1001 384-well Nucleofector Service Contract Valid for 1 year, can be purchased at any time during the guarantee period.Comprises diagnosis and repair of the system along <strong>with</strong> all replacement parts.AWT-1001 AWT-1001 384-well Nucleofector Installation and Training4Transfection / Nucleofector DevicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com175
Notes4Transfection / Nucleofector Devices176North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits4Nucleofector KitsNucleofector Kits for Primary Cells – Overview 178Nucleofector Kits for Primary Adipocytes 186Nucleofector Kits for Primary Blood Cells 187Nucleofector Kits for Primary Bone /Cartilage Cells 198Nucleofector Kits for Primary Cardiac Cells 199Nucleofector Kits for Primary Dermal Cells 200Nucleofector Kits for Primary Endothelial Cells 202Nucleofector Kits for Primary Epithelial Cells 206Nucleofector Kits for Fibroblasts 209Nucleofector Kits for Primary Hepatocytes 212Nucleofector Kits for Primary Muscle Cells 214Nucleofector Kits for Primary Neural Cells 217Nucleofector Kits for Primary Stem Cells 222Nucleofector Kits for Cell Lines 230Nucleofector Kits for Parasites 238Transfection / Nucleofector KitsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com177
Primary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsWith our new conductive polymer cuvette concept, whichwas first established for the 96-well Shuttle System andnow transferred to the new platforms, we were able tostreamline our kit concept for primary cells. For the4D-Nucleofector, 96-well Shuttle and 384-wellNucleofector Systems we now offer five different PrimaryCell Nucleofector Solutions P1, P2, P3, P4 and P5.■■Benefits––Five different Nucleofector Solutions – 1 NucleofectorKit can be used for multiple primary cell types––Conditions are transferable between 4D NucleofectorSystem, 96-well Shuttle System and 384-wellNucleofector System––Primary cells maintain functionality post transfection4Transfection / Nucleofector Kits for Primary Cells – Overview■■Each kit contains––Specific Nucleofector Solution––Supplement––pmaxGFP Control Vector––Either single 100 µl Nucleocuvettes, 16-wellNucleocuvette Strips, 96-well or 384-wellNucleocuvette PlatesAll kits are available in different package variations.Please refer to ordering information for details. OptimizedProtocols are available for download on our website. Inthese Optimized Protocols the best NucleofectionConditions are indicated. In addition, we share ourexperience and knowledge for treatment of individualprimary cell types. You can always find the most up-to-dateinformation in our online cell database.100 μl Nucleocuvette(4D-Nucleofector System)16-well Nucleocuvette Strip(4D-Nucleofector System)■■Applications––Transfection of lower cell numbers (from 2 × 10 4 to1 × 10 6 cells) and higher cell numbers (from 2 × 10 5 to2 × 10 7 cells) is possible––Flexible throughput from single cuvette (100 μl) to16-well Nucleocuvette Strip (20 μl), 96-well and384-well Nucleocuvette Plates is possiblewww.lonza.com/celldatabasewww.lonza.com/protocols96-well Nucleocuvette Plate(96-well Shuttle System)384-well Nucleocuvette Plate(384-well Nucleofector System)Ordering information on the next page.178North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Primary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsContinuedOrdering Information – KitsCat. No. Description Size4D-Nucleofector KitsV4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 12 rxn (100 µl Nucleocuvette)V4XP-1024 24 rxn (100 µl Nucleocuvette)V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 32 rxn (20 µl Nucleocuvette; 16-well)V4XP-2012 P2 Primary Cell 4D-Nucleofector X Kit L 12 rxn (100 µl Nucleocuvette)V4XP-2024 24 rxn (100 µl Nucleocuvette)V4XP-2032 P2 Primary Cell 4D-Nucleofector X Kit S 32 rxn (20 µl Nucleocuvette; 16-well)V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 12 rxn (100 µl Nucleocuvette)V4XP-3024 24 rxn (100 µl Nucleocuvette)V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 32 rxn (20 µl Nucleocuvette; 16-well)V4XP-4012 P4 Primary Cell 4D-Nucleofector X Kit L 12 rxn (100 µl Nucleocuvette)V4XP-4024 24 rxn (100 µl Nucleocuvette)V4XP-4032 P4 Primary Cell 4D-Nucleofector X Kit S 32 rxn (20 µl Nucleocuvette; 16-well)V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 12 rxn (100 µl Nucleocuvette)V4XP-5024 24 rxn (100 µl Nucleocuvette)V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 32 rxn (20 µl Nucleocuvette; 16-well)96-well Shuttle KitsV4SP-1096 P1 Primary Cell 96-well-Nucleofector Kit 96 rxn (20 µl Nucleocuvette; 96-well)V4SP-1960 960 rxn (20 µl Nucleocuvette; 96-well)V4SP-2096 P2 Primary Cell 96-well-Nucleofector Kit 96 rxn (20 µl Nucleocuvette; 96-well)V4SP-2960 960 rxn (20 µl Nucleocuvette; 96-well)V4SP-3096 P3 Primary Cell 96-well-Nucleofector Kit 96 rxn (20 µl Nucleocuvette; 96-well)V4SP-3960 960 rxn (20 µl Nucleocuvette; 96-well)V4SP-4096 P4 Primary Cell 96-well-Nucleofector Kit 96 rxn (20 µl Nucleocuvette; 96-well)V4SP-4960 960 rxn (20 µl Nucleocuvette; 96-well)V4SP-5096 P5 Primary Cell 96-well-Nucleofector Kit 96 rxn (20 µl Nucleocuvette; 96-well)V4SP-5960960 rxn (20 µl Nucleocuvette; 96-well)384-well Nucleofector KitsV5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 768 rxn (20 µl Nucleocuvette; 384-well)V5SP-1010 3840 rxn (20 µl Nucleocuvette; 384-well)V5SP-2002 P2 Primary Cell 384-well Nucleofector Kit 768 rxn (20 µl Nucleocuvette; 384-well)V5SP-2010 3840 rxn (20 µl Nucleocuvette; 384-well)V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 768 rxn (20 µl Nucleocuvette; 384-well)V5SP-3010 3840 rxn (20 µl Nucleocuvette; 384-well)V5SP-4002 P4 Primary Cell 384-well Nucleofector Kit 768 rxn (20 µl Nucleocuvette; 384-well)V5SP-4010 3840 rxn (20 µl Nucleocuvette; 384-well)V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 768 rxn (20 µl Nucleocuvette; 384-well)V5SP-50103840 rxn (20 µl Nucleocuvette; 384-well)4Transfection / Nucleofector Kits for Primary Cells – OverviewEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com179
Primary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsContinuedQuick Reference GuideKits for 4D-Nucleofector (Cat. No.) Kits for 96-well Shuttle (Cat. No.)Cell types Efficiency Viable cells Solution 100 µl (12 rxn)Cat. No.100 µl (24 rxn)Cat. No.20 µl (32 rxn)Cat. No.20 µl (96 rxn)Cat. No.20 µl (960 rxn)Cat. No.AdipocytesPre-adipocytes, human, visceral 37-94% 35-90% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960Pre-adipocytes, human, subcutaneous 51-84% 33-85% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960Pre-adipocytes, human, visceral28-65% 64-84% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960(Diabetes Type II)Pre-adipocytes, human, subcutaneous(Diabetes Type II)31-70% 61-95% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960Bone/Cartilage CellsChondrocyte, human 74% 84% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-39604Dermal CellsKeratinocyte, human, neonatal (NHEK) 60–70% 50–60% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Transfection / Nucleofector Kits for Primary Cells – OverviewEndothelial CellsEndothelial, aortic (HAEC), human 73% 70% P5 V4XP-5012 V4XP-5024 V4XP-5032 V4SP-5096 V4SP-5960Endothelial, microvascular, lung79% 48% P5 V4XP-5012 V4XP-5024 V4XP-5032 V4SP-5096 V4SP-5960(HMVEC-L), humanEndothelial, umbilical vn.(HUVEC), human 90% 55% P5 V4XP-5012 V4XP-5024 V4XP-5032 V4SP-5096 V4SP-5960Epithelial CellsEpithelial, bronchial (NHBE), human 54% 53% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Epithelial, bronchial, human, asthmatic 72% 75% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Epithelial, bronchial, human, COPD 63% 80% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Epithelial, mammary (HMEC), human 51% 66% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Epithelial, prostate (PrEC), human 67% 48% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960FibroblastsFibroblast, dermal (NHDF), human – adult 92–96% 92–100% P2 V4XP-2012 V4XP-2024 V4XP-2032 V4SP-2096 V4SP-2960Fibroblast, dermal (NHDF), human – neo 98% 86-91% P2 V4XP-2012 V4XP-2024 V4XP-2032 V4SP-2096 V4SP-2960Fibroblast , embryonic (MEF), mouse 68% 85-90% P4 V4XP-4012 V4XP-4024 V4XP-4032 V4SP-4096 V4SP-4960Hematopoietic CellsB cell, mouse, stimulated 55–56% 41–87% P4 V4XP-4012 V4XP-4024 V4XP-4032 V4SP-4096 V4SP-4960B cell, peripheral blood, CD19 + , human 28% 70% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Dendritic cell, mouse, mature – BALB/c 32% 85% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Dendritic cell, mouse, immat. – BALB/c 43% 37–49% P4 V4XP-4012 V4XP-4024 V4XP-4032 V4SP-4096 V4SP-4960Dendritic cell, mouse, mature – C57BL/6 29% 88% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Dendritic cell, mouse, immat. – C57BL/6 34% 41–58% P4 V4XP-4012 V4XP-4024 V4XP-4032 V4SP-4096 V4SP-4960T cell, human stimulated 70% 59% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960T cell, human unstimulated 69–87% 53–79% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960T cell, mouse – BALB/c 45% 32% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960T cell, mouse – C57BL/6 43% 23% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960180North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Primary Cell Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsContinuedQuick Reference GuideKits for 4D-Nucleofector (Cat. No.) Kits for 96-well Shuttle (Cat. No.)Cell types Efficiency Viable cells Solution 100 µl (12 rxn)Cat. No.100 µl (24 rxn)Cat. No.20 µl (32 rxn)Cat. No.20 µl (96 rxn)Cat. No.20 µl (960 rxn)Cat. No.Macrophage, human 42% 60% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Monocyte CD14 + , human 64% 77% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960HepatocytesHepatocyte, human 54% 59–69% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Muscle CellsSkeletal Muscle Myoblasts, human 72-78% 61% P5 V4XP-5012 V4XP-5024 V4XP-5032 V4SP-5096 V4SP-5960SMC, aortic (AoSMC), human 80% 53–80% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960Neural CellsNeuron, cortical, rat 30–50% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Neuron, hippocampal, rat 30–50% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-39604Stem CellsCD34 + cell, bone marrow, human 83% 62% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Embryonic stem (ES) cell, human 64% 98% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Embryonic stem (ES) cell, mouse 86–90% 68–81% P3 V4XP-3012 V4XP-3024 V4XP-3032 V4SP-3096 V4SP-3960Mesenchymal stem cells (MSC), human 69–78% 67–71% P1 V4XP-1012 V4XP-1024 V4XP-1032 V4SP-1096 V4SP-1960Transfection / Nucleofector Kits for Primary Cells – OverviewEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com181
Adherent Nucleofector Kits for 4D-Nucleofector SystemFor adherent Nucleofection using the 4D-NucleofectorY Unit, specific kits are required including an optimized24-well Dipping Electrode Array made <strong>with</strong> conductivepolymer electrodes.Following our new simplified kit strategy invented <strong>with</strong> the4D-Nucleofector System we offer two NucleofectorSolutions called AD1 and AD2, both available as separatekits or combined to an optimization kit. Each solution mayserve different cell types. You can easily find out whichsolution is optimal for your cell of interest by using thescheme on the right.4Transfection / Nucleofector Kits for Primary Cells – Overview■■Each kit contains––Specific Nucleofector Solution––Supplement––pmaxGFP Control Vector––24-well Dipping Electrode Array––Nunclon Δ Surface 24-well plate (Nunc)■■Benefits––Nucleofection of cells at any time point during thisculture period, i.e. at a later developmental stage––Transfection efficiencies up to 70% combined <strong>with</strong> highviabilities■■Applications––Two 4D-Nucleofector Y Kits that may serve differentcell types––An Optimization 4D-Nucleofector Y Kit for primary cellslacking an optimized protocolwww.lonza.com/celldatabaseNeurons or glial cells Endothelial cells Other cellsBasic protocolfor neuronsAD14D-Nucleofector Y KitBasic protocolfor endothelial cellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description SizeAdherent Nucleofection KitsV4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactionsV4YP-2A24 V4YP-2A24 AD2 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactionsV4YP-9A48 V4YP-9A48 Primary Cell Optimization 4D-Nucleofector Y Kit 24-well Dipping Electrode 48 reactionsOptimizationprotocolOptimization4D-Nucleofector Y KitAD24D-Nucleofector Y KitRelated ProductsPage4D-Nucleofector Y Unit 172182North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Primary Cell Optimization Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsThe new Primary Cell Optimization Nucleofector Kits arethe ideal tool to conveniently and rapidly determineNucleofection Conditions for primary cell types lacking anOptimized Protocol.Different conditions can easily be tested <strong>with</strong>in oneexperiment using any of the Nucleofector Platforms(4D-Nucleofector, 96-well Shuttle and 384-wellNucleofector System) as all of them are able to addressindividual wells of a 16-well, 96-well or 384-wellNucleocuvette Plate <strong>with</strong> different programs. In eachsystem our five Primary Cell Nucleofector SolutionsP1 – P5 are tested together <strong>with</strong> a pre-selected set ofprograms plus controls.■■Benefits––Convenient and rapid determination of optimalNucleofection Conditions for a broad range of primarycells <strong>with</strong>in one experiment––Optimal Nucleofection Conditions determined on oneplatform are transferable to the other platforms andalso to the 100 µl single Nucleocuvette in the4D-Nucleofector X Unit■■Applications––Determination of Nucleofection Conditions for primarycell types lacking an Optimized ProtocolPlatform 4D-Nucleofector System 96-well Shuttle System 384-well Nucleofector SystemNucleocuvette Vessel4Kit contents- Six 16-well Nucleocuvette Strips- Specific Nucleofector Solution- Supplement- pmaxGFP Control Vector- Two 96-well Nucleocuvette Plates- Specific Nucleofector Solution- Supplement- pmaxGFP Control VectorNumber of optimization reactions 80 rxn (plus 16 rxn for optional fine tuning) 160 rxn 384 rxnOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size- One 384-well Nucleocuvette Plate- Specific Nucleofector Solution- Supplement- pmaxGFP Control Vector4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II /2b KitsV4YP-9A48 V4YP-9A48 Primary Cell Optimization 4D-Nucleofector Y Kit 24-well Dipping Electrode 48 reactionsTransfection / Nucleofector Kits for Primary Cells – OverviewEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com183
Primary Cell Kits for Nucleofector II /2b DeviceThe Nucleofector II/2b uses cell type specific kits, each ofthem dedicated to an individual primary cell. Individuallydeveloped Nucleofector Kits are available for each primarycell type to use in combination <strong>with</strong> the Nucleofector II/2bDevice.■■Each Kit Contains––Specific Nucleofector Solution––Supplement––Single use pipettes––pmaxGFP Control Vector––Certified 100 µl aluminum electrode cuvettesAll Primary Cell Kits for the Nucleofector II/2b Device areavailable in different package variations (10, 25 and 4 × 25reactions) and include a CD containing all cell-type specificOptimized Protocols. The best Nucleofection Conditions areindicated in these optimized protocols. In addition we shareour experience and knowledge for treatment of individualprimary cell types. You can always find the most up-to-dateinformation in our online cell database.www.lonza.com/celldatabaseSee pages 186-2384Quick Reference GuideCell types Efficiency Viable cells 10 rxnCat. No.Kits for Nucleofector II/2b (Cat. No.)25 rxnCat. No.100 rxnCat. No.Transfection / Nucleofector Kits for Primary Cells – OverviewBone/Cartilage CellsChondrocyte, human 65% 60-70% VAPF-1001 VPF-1001 VVPF-1001Cardiac CellsCardiomyocyte, rat 75-80% 50-60% VAPE-1002 VPE-1002 VVPE-1002Dermal CellsKeratinocyte, adult (NHEK-Ad), human 51% 40-60% VAPD-1002 VPD-1002 VVPD-1002Keratinocyte, neonatal (NHEK-neo), human 39-53% 50-60% VAPD-1002 VPD-1002 VVPD-1002Melanocyte, neonatal (NHEM-neo), human 70% 55-60% VAPD-1003 VPD-1003 VVPD-1003Endothelial CellsEndothelial, coronary artery (HCAEC), human 57% 42% VAPB-1001 VPB-1001 VVPB-1001Endothelial, microvascular, lung (HMVEC-L), human 52% 52% VAPB-1003 VPB-1003 VVPB-1003Endothelial, umbilical vein (HUVEC), human 90% 60-74% VAPB-1002 VPB-1002 VVPB-1002Epithelial CellsEpithelial, bronchial (NHBE), human 50-65% 50% VAPK-1001 VPK-1001 VVPK-1001Epithelial, mammary (HMEC), human 73% 66-98% VAPK-1002 VPK-1002 VVPK-1002Epithelial, prostate (PrEC), human 43% 64% VAPK-1003 VPK-1003 VVPK-1003FibroblastsEmbryonic fibroblast (MEF), mouse 43% 60-80% VPD-1006*Fibroblast, dermal (NHDF), human – adult 42-69% 74-77% VAPD-1001 VPD-1001 VVPD-1001Fibroblast, dermal (NHDF), human – neo 90% 85-90% VAPD-1001 VPD-1001 VVPD-1001*Starter Kit: different reaction size.Hematopoietic CellsB cell, peripheral blood, CD19 + , human 36% 84-92% VAPA-1001 VPA-1001 VVPA-1001B cell, mouse, stimulated 59% 27-47% VAPA-1010 VPA-1010 VVPA-1010Dendritic cell, human 93-99% 12-75% VAPA-1004 VPA-1004 VVPA-1004Dendritic cell, mouse, immature – BALB/c 58% 62% VAPA-1011 VPA-1011 VVPA-1011184North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Primary Cell Kits for Nucleofector II /2b DeviceContinuedQuick Reference GuideCell types Efficiency Viable cells 10 rxnCat. No.Kits for Nucleofector II/2b (Cat. No.)25 rxnCat. No.100 rxnCat. No.Dendritic cell, mouse, immature – C57BL/6 54% 52% VAPA-1011 VPA-1011 VVPA-1011Dendritic cell, mouse, mature – BALB/c 49% 78% VAPA-1011 VPA-1011 VVPA-1011Dendritic cell, mouse, mature – C57BL/6 37% 63% VAPA-1011 VPA-1011 VVPA-1011Macrophage, human 55-59% 87-88% VAPA-1008 VPA-1008 VVPA-1008Macrophage, mouse – BALB/c 34-45% 84-92% VAPA-1009 VPA-1009 VVPA-1009Macrophage, mouse – C57BL/6 24-47% 80-88% VAPA-1009 VPA-1009 VVPA-1009Monocyte CD14 + , human 60% 62-81% VAPA-1007 VPA-1007 VVPA-1007Natural killer (NK), human 54% 50-60% VAPA-1005 VPA-1005 VVPA-1005T cell, human stimulated 41-47% 83-90% VAPA-1002 VPA-1002 VVPA-1002T cell, human unstimulated 70-75% 85% VAPA-1002 VPA-1002 VVPA-1002T cell, mouse – BALB/c 44% 18-55% VAPA-1006 VPA-1006 VVPA-1006T cell, mouse – C57BL/6 20-28% 17-45% VAPA-1006 VPA-1006 VVPA-1006HepatocytesHepatocyte, mouse 54% 80% VAPL-1002 VPL-1002 VVPL-1002Hepatocyte, rat 52% 78% VAPL-1003 VPL-1003 VVPL-1003Neural CellsAstrocyte, mixed brain, C57 mouse 60% 60-70% VAPI-1006 VPI-1006 VVPI-1006Astrocyte, mixed brain, CD1 mouse 60% 60-70% VAPI-1006 VPI-1006 VVPI-1006Astrocytes, striatum,rat 67% 70-80% VAPI-1006 VPI-1006 VVPI-1006Dorsal root ganglia (DRG), rat 41% VAPG-1003 VPG-1003 VVPG-1003Dorsal root ganglia (DRG), chicken 30% VAPG-1002 VPG-1002 VVPG-1002Neuron, cortical, rat 58-67% 47-60% VAPG-1003 VPG-1003 VVPG-1003Neuron, hippocampal, chicken 43% VAPG-1002 VPG-1002 VVPG-1002Neuron, hippocampal, rat 58-67% 47-60% VAPG-1003 VPG-1003 VVPG-1003Neuron, hippocampal, mouse 58% VAPG-1001 VPG-1001 VVPG-1001Oligodendrocyte, rat 44% 60% VAPI-1006 VPI-1006 VVPI-1006Smooth Muscle CellsSmooth muscle cell, aortic (AoSMC), human 75% 69-96% VAPC-1001 VPC-1001 VVPC-1001Stem CellsCD34 + cell, bone marrow, human 82% 70% VAPA-1003 VPA-1003 VVPA-1003Embryonic stem (ES) cell, human 20-78% 50-96% VPH-5002*Embryonic stem (ES) cell, mouse 87-90% 90-99% VAPH-1001 VPH-1001 VVPH-1001Mesenchymal stem cell (MSC), human 55-88% 50-86% VAPE-1001 VPE-1001 VVPE-1001Neural stem cell (NSC), mouse 82% VAPG-1004 VPG-1004 VVPG-1004Neural stem cell (NSC), rat 42-46% VAPG-1005 VPG-1005 VVPG-1005*Starter Kit: different reaction size4Transfection / Nucleofector Kits for Primary Cells – OverviewEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com185
Nucleofector Kits for Human Pre-AdipocytesOptimal kits for transfection of human pre-adipocytes in the4D-Nucleofector X Unit are the P1 Primary Cell Kits, used incombination <strong>with</strong> the cell-type specific protocol. Due totransferability between all platforms, same conditionsapply for the 96-well Shuttle or 384-well NucleofectorSystems.AB4Transfection / Nucleofector Kits for Primary Adipocytes■■Benefits––Transfection efficiency: up to 90%––Viability: up to 80%––Cells can be differentiated into adipocytes postNucleofection■■Applications––Validated to work <strong>with</strong> visceral and subcutaneousPoietics Human Preadipocytes––Also tested <strong>with</strong> Diabetes Type II pre-adipocytes––Easily verify previous cell line results in the analogousprimary cell typeExample of Nucleofection of human pre-adipocytes. Poietics HumanVisceral Preadipocytes were transfected <strong>with</strong> pmaxGFP Vector anddifferentiated into adipocytes post Nucleofection using PGM-2 AdipocyteDifferentiation Medium. After 14 days cells were analyzed by AdipoRedAssay (A: Non-transfected control; B: transfected cells). Quantitativeanalysis showed that more than 80% of transfected sample stainedpositive for AdipoRed (normalized to non-transfected control set to 100%).Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-1012 V4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-1024 V4XP-1024 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-1032 V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-1960 V4SP-1960 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SP-1096 V4SP-1096 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)384-well Nucleofector KitsV5SP-1010 V5SP-1010 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SP-1002 V5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)Related ProductsPageHuman Preadipocyte Cells, normal or diseased 85PGM-2 Preadipocyte Growth Medium-2 BulletKit 86AdipoRed Assay Reagent 259186North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human B CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of human B cellsusing the different Nucleofection Platforms.Optimal kits for transfection of human B cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Human B cellspecific kits are available for the Nucleofector II/2b Device.% CAT activity400350300250200150100■■Benefits––Transfection efficiency: up to 36% using a non-viralmethod––Viability: up to 92%■■Applications––Kits suitable for human CD19 + B cells from peripheralblood––Also applicable for transfection of CLL cells derived frompatient material––For both unstimulated and stimulated B cells––Same conditions for DNA, siRNA, or RNA500CR2CR2 + p50 CR2 + p65ControlPromoter studies in primary B cells prove that NF-KB regulates the activityof the human CR2 promoter. Primary human B cells were transientlyco-transfected <strong>with</strong> a CAT reporter plasmid driven by wild-type (WT) CR2promoter, and plasmids encoding NF-KB subunit p50 or p65 or a controlplasmid. Cells were assayed for CAT activity 15 hours post Nucleofection.Values represent percentage of CAT activity, considering the activity of theempty vector control 0% and the activity of the wild-type CR2 promoter100%. The results demonstrate that both NF-KB subunits clearly induce CATactivity. (Data reproduced from Tolnay et al. (2002) J Immunology, <strong>with</strong>permission from the Journal of Immunology.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1001 VAPA-1001 Human B Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1001 VPA-1001 Human B Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1001 VVPA-1001 Human B Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageRPMI 1640 <strong>with</strong>out L-glutamine 110LGM-3 Lymphocyte Growth Medium-3 93-95X-VIVO 20 Chemically Defined, Serum-free Hematopoietic Cell Medium 1204Transfection / Nucleofector Kits for Primary Blood CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com187
Nucleofector Kits for Stimulated Mouse B CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of stimulatedmouse B cells using the different Nucleofection Platforms.Optimal kits for transfection of mouse B cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P4 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Mouse B cellspecific kits are available for the Nucleofector II/2b Device.AB■■Benefits––Transfection efficiency: up to 59%––Viability: up to 87%––Expression of cell typical marker proteins not affectedNucleofection of mouse B cells. Primary mouse B cells were transfectedby Nucleofection using a plasmid encoding maxGFP Reporter Protein.Cells were then stimulated <strong>with</strong> LPS. 48 hours post Nucleofection, cellswere analyzed by light (A) and fluorescence microscopy (B).4■■Applications––Kits suitable for stimulated mouse B cells––Same conditions for DNA, siRNA and RNA transfectionTransfection / Nucleofector Kits for Primary Blood CellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-4012 V4XP-4012 P4 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-4024 V4XP-4024 P4 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-4032 V4XP-4032 P4 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-4096 V4SP-4096 P4 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-4960 V4SP-4960 P4 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-4002 V5SP-4002 P4 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-4010 V5SP-4010 P4 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1010 VAPA-1010 Mouse B Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1010 VPA-1010 Mouse B Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1010 VVPA-1010 Mouse B Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageRPMI 1640 <strong>with</strong>out L-glutamine 110188North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Dendritic CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humandendritic cells using the different Nucleofection Platforms.For the transfection of human dendritic cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Humandendritic cell specific kits are available for theNucleofector II /2b Device.■■Benefits––Transfection efficiency: up to 49%––Viability: up to 71%––Same transfection conditions <strong>with</strong> different substrates,such as RNA, DNA or siRNA■■Applications––Kit suitable for immature and mature monocyte-deriveddendritic cells––For short term expression of up to 48 hours% Firefly luciferase expression*% Viability12011010090807060504030201001201101009080706050403020100nono100 scr100 scr100 luc100 luc500 scr500 scr500 luc500 lucCo-transfection of human DCs <strong>with</strong> plasmid and siRNA. Cells weretransfected by Nucleofection <strong>with</strong> a CMV promoter driven firefly luciferasevector (pCMV-Luc), TK-promoter driven Renilla luciferase vector (pTK-Luc)as internal control reporter for normalization, and siRNA against fireflyluciferase (luc) or scrambled control (scr). 24 hours post Nucleofection,cells were analyzed for luciferase activity. Viability was determined by anATP-content assay. (Data reproduced from Stallwood et al. (2006) JImmunol 177(2):885-895, <strong>with</strong> permission of the authors).*Normalized to Renilla luciferase as internal control reporterOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II /2b KitsVAPA-1004 VAPA-1004 Human Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1004 VPA-1004 Human Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1004 VVPA-1004 Human Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactions4Transfection / Nucleofector Kits for Primary Blood CellsRelated ProductsPageRPMI 1640 <strong>with</strong>out L-glutamine 110LGM-3 Lymphocyte Growth Medium-3 93-95Normal Human Dendritic Cells 93Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com189
Nucleofector Kits for Mouse Dendritic Cells4Transfection / Nucleofector Kits for Primary Blood CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mousedendritic cells (DCs) using the different NucleofectionPlatforms.Optimal kits for transfection of mouse dendritic cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits (formature DCs) and P4 Primary Cell Kits (for immature DCs)used in combination <strong>with</strong> cell-type specific protocols.Mouse dendritic cell specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 58% using a non-viralmethod––Viability: up to 88%■■Applications––Kits suitable for Balb/C or C57BL/6 mouse DCs––Proven results for immature and mature mouse DCs––Ideal for gene over-expression studies or RNAi mediatedgene silencingAFunctioanilty1101009080706050403020100ControlSampleTransfection efficiency and functionality of mouse DCs postNucleofection. (A) The graph displays functionality of immature mouseDCs (isolated from mouse strain Balb/C) post Nucleofection (Sample).Two hours post Nucleofection, cells were stimulated by LPS. 22 hourslater, functionality was analyzed by IL-6 specific ELISA and is given inpercent compared to non-transfected control. (B) Mouse DC (Balb/C) weretransfected using pmaxGFP Vector. Cells were analyzed 24 hours postNucleofection by flow cytometry for maxGFP Reporter Protein expressionand viability. Cell viability is given in percent compared to non-transfectedcontrol.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L* 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L* 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S* 20 μl Nucleocuvette 32 reactions (16-well)V4XP-4012 V4XP-4012 P4 Primary Cell 4D-Nucleofector X Kit L** 100 μl Nucleocuvette 12 reactionsV4XP-4024 V4XP-4024 P4 Primary Cell 4D-Nucleofector X Kit L** 100 μl Nucleocuvette 24 reactionsV4XP-4032 V4XP-4032 P4 Primary Cell 4D-Nucleofector X Kit S** 20 μl Nucleocuvette 32 reactions (16-well)B% Transfection efficiency1101009080706050403020100Transfection efficiencyViability96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit* 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit* 20 μl Nucleocuvette 960 reactions (96-well)V4SP-4096 V4SP-4096 P4 Primary Cell 96-well Nucleofector Kit** 20 μl Nucleocuvette 96 reactions (96-well)V4SP-4960 V4SP-4960 P4 Primary Cell 96-well Nucleofector Kit** 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit* 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit* 20 μl Nucleocuvette 3840 reactions (384-well)V5SP-4002 V5SP-4002 P4 Primary Cell 384-well Nucleofector Kit** 20 μl Nucleocuvette 768 reactions (384-well)V5SP-4010 V5SP-4010 P4 Primary Cell 384-well Nucleofector Kit** 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1011 VAPA-1011 Mouse Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1011 VPA-1011 Mouse Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1011 VVPA-1011 Mouse Dendritic Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactions*For mature Mouse DCs **For immature Mouse DCs190North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human MacrophagesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanmacrophages using the different Nucleofection Platforms.Optimal kits for transfection of human macrophages in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Humanmacrophage specific kits are available for the NucleofectorII/2b Device.■■Benefits––Transfection efficiency: up to 59%––Viability: up to 88%––Maintenance of functionality (e.g. activation)ACBD■■Applications––Kits suitable for resting human macrophages––Cited for DNA and siRNA transfection––High-throughput screening approaches possibleNucleofection of human macrophages. Primary human macrophageswere transfected by Nucleofection <strong>with</strong> pmaxGFP Control Vector. 24hours post Nucleofection, cells were analyzed for maxGFP ReporterProtein expression by light (A, C) and fluorescence (B, D) microscopy. Aand B show cells at 10x magnification. At 40x magnification (C, D)transfected macrophages reveal cytoplasmatic extrusions important forphagocytic function of macrophages.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1008 VAPA-1008 Human Macrophage Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1008 VPA-1008 Human Macrophage Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1008 VVPA-1008 Human Macrophage Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactions4Transfection / Nucleofector Kits for Primary Blood CellsRelated ProductsRPMI 1640 <strong>with</strong>out L-glutamine 110PageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com191
Nucleofector Kits for Mouse MacrophagesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mousemacrophages using the different Nucleofection Platforms.For the transfection of mouse macrophages in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Mousemacrophage specific kits are available for the NucleofectorII/2b Device.ACBD4■■Benefits––Transfection efficiency: up to 47%––Viability: up to 92%––Maintenance of functionality (e.g., activation)Transfection / Nucleofector Kits for Primary Blood Cells■■Applications––Kits suitable for resting bone marrow-derived mousemacrophages––Evaluated for C57BL/6 and BALB/c strains––Enabling studies of gene regulation, signaling pathwaysor differentiationRelated ProductsNucleofection of mouse macrophages <strong>with</strong> pmaxGFP Vector. Primarymouse macrophages were transfected by Nucleofection <strong>with</strong> a plasmidencoding maxGFP Reporter Protein. 24 hours post Nucleofection, cellswere analyzed by light (A, C) and fluorescence microscopy (B, D). A and Bshow cells at 10 × magnification. At 40 × magnification (C, D), transfectedmacrophages reveal cytoplasmic extrusions important for phagocyticfunction.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPA-1009 VAPA-1009 Mouse Macrophage Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1009 VPA-1009 Mouse Macrophage Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1009 VVPA-1009 Mouse Macrophage Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsDMEM 4.5 g/L glucose <strong>with</strong> L-glutamine 103RPMI 1640 <strong>with</strong>out L-glutamine 110Page192North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human MonocytesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanmonocytes using the different Nucleofection Platforms.Optimal kits for transfection of human monocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Humanmonocyte specific kits are available for the NucleofectorII/2b Device.■■Benefits––Transfection efficiency: up to 64%––Viability: up to 81%––First high-throughput transfection technology forhuman monocytes■■Applications––Kits suitable for CD14 + human monocytes––Cited for DNA and siRNA transfections% CX3CR1 + cells010 10 1 10 2 10 3 10 4FL1-H50454035302520151050None9-HODELipidControl PPAR γ #1Nucleofection of human monocytes <strong>with</strong> Stealth siRNA. (A) Efficiency oftransfection was determined <strong>with</strong> 100 nM fluorescein-labeled dsRNAoligomer (same length, electrical charge and configuration as the siRNA)monitored 24 hours later by flow cytometry. Blue curve showsautofluorescence. (B, C and D) Knockdown <strong>with</strong> Stealth siRNA(Invitrogen). Oxidized linoleic acid metabolites (like 9-HODE,9-hydroxy-10E, 12Z-octadecadienoic acid ester), components of oxidizedLDL found in large amounts in atherosclerotic plaque, are able tospecifically induce differentiation of human monocytes to macrophagesaccompanied by a switch of chemokine receptor expression (CCR2-off andCX3CR1-on). CX3CR1 then mediates macrophage adhesion to coronaryarter y smooth muscle cells (CASMCs). The effects of the lipids on receptorexpression are mediated by the nuclear receptor peroxisome proliferatoractivatedreceptor (PPAR) γ. Down regulation of PPARγ <strong>with</strong> siRNA(200 nM, (Invitrogen)) dramatically reduced receptor switch (B and C)and consequently macrophage adhesion to CASMCs in an adhesion assay(D). (Data extracted from Barlic et al., (2006) Circulation 114(8), 807-19<strong>with</strong> permission from the authors.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsCounts2402001601208040–Adhesion (endpointfluoesence U/ml 10-6)% CCR2 + cells141210864202520151050– ControlsiRNAiNo stimulusV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVPA-1007 VPA-1007 Human Monocyte Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1007 VVPA-1007 Human Monocyte Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsAB98.73%DC–NoneLipidControlPPAR γ #19-HODE9-HODEPPAR γ #14Transfection / Nucleofector Kits for Primary Blood CellsRelated ProductsPageLGM-3 Lymphocyte Growth Medium-3 93-95Human CD14 + Monocytes 94Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com193
Nucleofector Kits for Human Natural Killer Cells4Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of human NKcells using the different Nucleofection Platforms.For the transfection of human NK cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Human NK cellspecific kits are available for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 54%––Viability: up to 60%––Efficient non-viral transfection technology for primaryNK cellsSSCANK cellseGFP-DNASSCNK cellsNucleofection of primary human NK cells. Polyclonal human NK cellsgenerated from PBMC co-cultured <strong>with</strong> the feeder cell line RPMI 8866 for 9days were transfected by Nucleofection <strong>with</strong> a plasmid encoding eGFPprotein. Cells were analyzed by flow cytometry 24 hours postNucleofection. eGFP expression in natural killer cells is shown afterNucleofection <strong>with</strong>out (A) and <strong>with</strong> plasmid DNA (B). (Courtesy of J.Sundback and K. Karre, Karolinska Institute, Microbiology and TumorBiology Center, Stockholm, Sweden.)B+DNA0.5% 16.9%eGFPTransfection / Nucleofector Kits for Primary Blood Cells■■Applications––Kits suitable for human CD56 + /CD3 – natural killer cellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPA-1005 VAPA-1005 Human Natural Killer Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1005 VPA-1005 Human Natural Killer Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1005 VVPA-1005 Human Natural Killer Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsLGM-3 Lymphocyte Growth Medium-3 93-95RPMI 1640 <strong>with</strong>out L-glutamine 110Human NK Cells 95Page194North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human T CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of human T cellsusing the different Nucleofection Platforms.Optimal kits for transfection of human T cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Human T cellspecific kits are available for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 87%––Viability: up to 90%––Transfected cells preserve their biochemicalfunctionality––More than 270 publications on T cell Nucleofection■■Applications––Kits suitable for stimulated and unstimulated humanT cells––RNAi screenings in primary T cells for basic andpharmaceutical researchwww.lonza.com/citationsCD3-APC side scatterCD3-APCforward scatter FL1 FL2- DNA+DNA0.14% 60.64%FL1FL1Nucleofection of human T cells <strong>with</strong> pmaxGFP Vector. PBMC were freshlyisolated from a buffy coat and transfected by Nucleofection <strong>with</strong>pmaxGFP Vector. 24 hours post Nucleofection, cells were analyzed byflow cytometry. Lymphocytes were gated according to forward/sidescatter (A). T cells were stained <strong>with</strong> antibody directed against CD3. Deadcells were excluded by propidium iodide staining and gating (B, C).maxGFP Reporter Protein expression of T cells is shown afterNucleofection <strong>with</strong>out (D) and <strong>with</strong> plasmid DNA (E).Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1002 VAPA-1002 Human T Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1002 VPA-1002 Human T Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1002 VVPA-1002 Human T Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsADBCD3-APCEPJC4Transfection / Nucleofector Kits for Primary Blood CellsRelated ProductsPageLGM-3 Lymphocyte Growth Medium-3 93-95IMDM <strong>with</strong> HEPES and L-glutamine 106Human CD4 + T Cells 95Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com195
Nucleofector Kits for Mouse T cells4Transfection / Nucleofector Kits for Primary Blood CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mouse T cellsusing the different Nucleofection Platforms.Optimal kits for transfection of mouse T cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Mouse T cellspecific kits are available for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 45%––Viability: up to 55%––Evaluated for C57BL/6 and BALB/c strains––Maintenance of functionality, e.g. stimulation■■Applications––Kits suitable for mouse T cells from C57BL/6 or BALB/c––Overexpression or gene silencing studies possible inhigh-throughput frameworks% CD25 expressing CD4 + cells1101009080706050403020100- Nucleofection/-DNA+ Nucleofection/-DNA+ Nucleofection/+DNA48 hours 72 hoursTransfected and non-transfected mouse T cells can be stimulated equallywell. Primary C57BL/6 mouse T cells were transfected using Nucleofection<strong>with</strong> pmaxGFP Vector. 3 hours post Nucleofection, cells were stimulated<strong>with</strong> plate bound anti-CD3 and anti-CD28. 48 and 72 hours postNucleofection, CD4 + cells were analyzed for CD25 surface expression.Figure shows proportion of CD25-expressing cells among living CD4 + Tcells (%CD25 expression in unstimulated samples ranged from 10–20%).Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVPA-1006 VPA-1006 Mouse T Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1006 VVPA-1006 Mouse T Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageMouse T Cell Nucleofector Medium 242196North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mammalian Blood CellsFor mammalian blood cells lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The Primary Cell Optimization Kits are suited foroptimizations of mammalian blood cells on the4D-Nucleofector System, the 96-well Shuttle System orthe 384-well Nucleofector System.■■Benefits––Protocols guiding through the optimization procedure––Optimizations can be performed <strong>with</strong>in one experiment––Optional result fine tuning <strong>with</strong> help from our ScientificSupport Team■■Applications––Kits suited for blood cells from different mammalianspecies and various organs4Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Transfection / Nucleofector Kits for Primary Blood CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com197
Nucleofector Kits for Human Chondroycytes4Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanchondrocytes using the different Nucleofection Platforms.Optimal kits for transfection of human chondrocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Humanchondrocyte cell specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 74%––Viability: up to 84%––First efficient non-viral transfection technology■■Applications––Optimal for studies of degenerative processes, such asosteoarthritisExample of the transfection of human chondrocytes <strong>with</strong> eGFP. Humanchondrocytes were transfected by Nucleofection using a plasmidencoding the enhanced green fluorescent protein eGFP. Cell membraneswere fluorescently stained in red <strong>with</strong> the substance R18(Octadecylrhodamine-B-chloride, Molecular Probes). 24 hours postNucleofection, the cells were analyzed by fluorescence microscopy. Theimage shows an overlay of eGFP and R18 fluorescence. (Data courtesy ofDr. Schmid and Dr. Aigner, University of Erlangen, Germany.)Transfection / Nucleofector Kits for Primary Bone/Cartilage CellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPF-1001 VAPF-1001 Human Chondrocyte Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPF-1001 VPF-1001 Human Chondrocyte Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPF-1001 VVPF-1001 Human Chondrocyte Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactions198North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Rat CardiomyocytesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of ratcardiomyocytes using the different NucleofectionPlatforms.For the transfection of rat cardiomyocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Ratcardiomyocyte specific kits are available for theNucleofector II/2b Device.ACB■■Benefits––Transfection efficiency: up to 80%––Viability: up to 60%––First efficient non-viral transfection technology4■■Applications––Kit suitable for neonatal rat cardiomyocytes––Optimal for studies of cardiac gene regulation anddifferentiationRelated ProductsExample for Nucleofection of neonatal rat cardiomyocytes <strong>with</strong> DsRed2cDNA. Primary neonatal rat cardiomyocytes were transfected byNucleofection using a plasmid encoding DsRed (Clontech). 2 days postNucleofection, the cells were analyzed by fluorescence microscopy. Fig.(A) shows DsRed expressing cells. Cardiomyocytes stained <strong>with</strong> FITClabeledtropomyosin antibody are shown in Fig. (B). Fig. (C) is an overlayof images (A) and (B). (Photograph courtesy of F. Engel and M. Keating,Cardiology Department, Children’s Hospital, Havard Medical School, Boston,Massachusetts, USA.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPE-1002 VAPE-1002 Rat Cardiomyocyte - Neonatal Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPE-1002 VPE-1002 Rat Cardiomyocyte - Neonatal Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPE-1002 VVPE-1002 Rat Cardiomyocyte - Neonatal Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRat Cardiac Myocytes 65–66RCGM – Rat Cardiac Growth Myocytes BulletKit 66PageTransfection / Nucleofector Kits for Primary Cardiac CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com199
Nucleofector Kits for Human Keratinocytes (NHEK)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humankeratinocytes using the different Nucleofection Platforms.Optimal kits for transfection of human keratinocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Humankeratinocyte specific kits are available for the NucleofectorII/2b Device.AB4Transfection / Nucleofector Kits for Primary Dermal Cells■■Benefits––Transfection efficiency: up to 53%––Viability: up to 60%––Maintenance of functionality, e.g. no terminaldifferentiation■■Applications––Validated to work <strong>with</strong> Clonetics Human Keratinocytes––Kits suitable for adult and neonatal keratinocytes––Optimal for studying gene expression or intracellularsignaling––Cited for DNA and siRNA transfectionsExample for the Nucleofection of human keratinocytes. CloneticsNHEK-neo were transfected by Nucleofection <strong>with</strong> pmaxGFP Vector. 48hours post Nucleofection, cells were analyzed by light (A) andfluorescence microscopy (B).www.lonza.com/citationsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPD-1002 VAPD-1002 Human Keratinocyte Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPD-1002 VPD-1002 Human Keratinocyte Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPD-1002 VVPD-1002 Human Keratinocyte Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageNHEK – Adult Normal Human Epidermal Keratinocytes 35NHEK – Neonatal Normal Human Epidermal Keratinocytes 35KGM-Gold Keratinocyte Growth Medium BulletKit 36200North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Melanocytes (NHEM-Neo)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanmelanocytes using the different Nucleofection Platforms.For the transfection of human melanocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Humanmelanocyte specific kits are available for the NucleofectorII/2b Device.■■Benefits––Transfection efficiency: up to 70%––Viability: up to 60%––Reproducible non-viral transfectionANucleofection of NHEM-Neo <strong>with</strong> eGFP cDNA. NHEM-Neo were transfectedby Nucleofection using a plasmid encoding enhanced green fluorescentprotein, eGFP. 24 hours post Nucleofection, cells were analyzed by light(A) and fluorescence microscopy (B).B4■■Applications––Kits suitable for neonatal human melanocytes(NHEM-neo)––Optimal for both DNA and siRNA transfectionOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPD-1003 VAPD-1003 Normal Human Epidermal Melanocyte - Neonatal Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPD-1003 VPD-1003 Human Melanocytes - Neonatal Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPD-1003 VVPD-1003 Human Epidermal Melanocyte - Neonatal Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageNHEM-Neo – Neonatal Normal Human Epidermal Melanocytes 35MGM-4 Melanocyte Growth Medium-4 BulletKit 36Transfection / Nucleofector Kits for Primary Dermal CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com201
Nucleofector Kits for Human Coronary Artery Endothelial Cells (HCAEC)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of HCAECs usingthe different Nucleofection Platforms.Optimal kits for transfection of HCAECs in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P5 Primary Cell Kits used incombination <strong>with</strong> respective basic protocols for mammalianendothelial cells. HCAEC specific kits are available for theNucleofector II/2b Device.AB■■Benefits––Transfection efficiency: up to 57%––Viability: up to 42%Example for the Nucleofection of HCAEC. Clonetics HCAEC weretransfected by Nucleofection <strong>with</strong> a plasmid encoding the fluorescentprotein eGFP. 25 hours post Nucleofection, cells were analyzed by light(A) and fluorescence microscopy (B).4■■Applications––Validated to work <strong>with</strong> Clonetics HCAEC––Ideal for cardiovascular research e.g., on thrombosis,atherosclerosis or hypertensionTransfection / Nucleofector Kits for Primary Endothelial CellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-5012 V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-5024 V4XP-5024 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-5032 V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-5096 V4SP-5096 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-5960 V4SP-5960 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-5002 V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-5010 V5SP-5010 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPB-1001 VAPB-1001 Human Coronary Artery Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPB-1001 VPB-1001 Human Coronary Artery Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPB-1001 VVPB-1001 Human Coronary Artery Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsHCAEC – Human Coronary Artery Endothelial Cells 32,38EGM-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit 30Page202North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Microvascular Endothelial Cells – Lung (HMVEC-L)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of HMVEC-Lusing the different Nucleofection Platforms.Optimal kits for transfection of HMVEC-L in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P5 Primary Cell Kits used incombination <strong>with</strong> respective basic protocols for mammalianendothelial cells. HMVEC-L specific kits are available for theNucleofector II/2b Device.AB■■Benefits––Transfection efficiency: up to 79%––Viability: up to 52%––Efficient transfection of HMVEC-L <strong>with</strong>out the use of aviral system■■Applications––Validated to work <strong>with</strong> Clonetics HMVEC-LExample for the Nucleofection of Clonetics HMVEC-L. HMVEC-L weretransfected by Nucleofection using a plasmid encoding the enhancedgreen fluorescent protein eGFP. 25 hours post Nucleofection, cells wereanalyzed by light (A) and fluorescence microscopy (B).4Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-5012 V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-5024 V4XP-5024 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-5032 V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-5096 V4SP-5096 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-5960 V4SP-5960 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-5002 V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-5010 V5SP-5010 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPB-1003 VAPB-1003 Human Microvascular Endothelial Cell-Lung Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPB-1003 VPB-1003 Human Microvascular Endothelial Cell-Lung Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPB-1003 VVPB-1003 Human Microvascular Endothelial Cell-Lung Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsHMVEC-L – Human Microvascular Endothelial Cells – Lung 40,52EGM-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit 30PageTransfection / Nucleofector Kits for Primary Endothelial CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com203
Nucleofector Kits for Human Umbilical Vein Endothelial Cells (HUVEC)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of HUVECs usingthe different Nucleofection Platforms.Optimal kits for transfection of HUVECs in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P5 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. HUVECspecific kits are available for the Nucleofector II/2b Device.Chemiluminescence ( x10 4 cpm)3.53.02.52.01.51.0NADHSOD4Transfection / Nucleofector Kits for Primary Endothelial Cells■■Benefits––Transfection efficiency: up to 90%––Viability: up to 74%––High protein expression levels possible––More than 60 publications on HUVEC Nucleofection■■Applications––Validated to work <strong>with</strong> Clonetics HUVEC––Ideal for siRNA screening in drug discovery projectsNucleofection in adherent state is possible using theAD1 4D-Nucleofector Y Kit0.50.0Time [min]- siRNA+ siRNANucleofection of HUVECs <strong>with</strong> siRNA. Knockdown of the NAD(P)H oxidaseNox4 <strong>with</strong> siRNA shows that Nox4 is the major source for superoxideproduction in the nucleus of HUVECs. 48 hours after Nucleofection ofHUVECs <strong>with</strong> Nox4 siRNA, the nuclear fraction was prepared and superoxideproduction was determined as superoxide dismutase (SOD)-inhibitablechemiluminescence detected <strong>with</strong> a luminol-based test. The reaction wasstarted by the addition of NADH and stopped by addition of SOD. (Data fromKuroda et al. (2005) Genes Cells 10(12), 1139-1151.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-5012 V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-5024 V4XP-5024 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-5032 V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-5096 V4SP-5096 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-5960 V4SP-5960 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-5002 V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-5010 V5SP-5010 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPB-1002 VAPB-1002 Human Umbilical Vein Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPB-1002 VPB-1002 Human Umbilical Vein Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPB-1002 VVPB-1002 Human Umbilical Vein Endothelial Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsHUVEC – Human Umbilical Vein Endothelial Cells 38EGM-2 Endothelial Cell Growth Medium-2 BulletKit 33,38,52102030Page204North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mammalian Endothelial CellsFor mammalian endothelial cells lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The P5 Primary Cell Kits together <strong>with</strong> the cell group-specificBasic Protocols are suited for optimizations of mammalianendothelial cells on the 4D-Nucleofector System, the96-well Shuttle System or the 384-well NucleofectorSystem.A cell-group specific Basic Kit is suited for optimization ofmammalian endothelial cells using the Nucleofector II/2bDevice.■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols provide guidance through theoptimization procedure––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team––Transfection efficiency: up to 90%––Viability: up to 85%Related Products■■Applications––Kits suited for endothelial cells from differentmammalian species and various organs––Already tested for human pulmonary artery endothelialcells (Clonetics HPAEC), porcine capillary endothelialcells, sheep uterine artery endothelial cells, etcNucleofection in adherent state is possible using theAD1 4D-Nucleofector Y KitOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-5012 V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-5024 V4XP-5024 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-5032 V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-5096 V4SP-5096 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-5960 V4SP-5960 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-5002 V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-5010 V5SP-5010 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1001 VAPI-1001 Basic Nucleofector Kit for Primary Mammalian Endothelial Cells 100 μl aluminum cuvette 10 reactionsVPI-1001 VPI-1001 Basic Nucleofector Kit for Primary Mammalian Endothelial Cells 100 μl aluminum cuvette 25 reactionsVVPI-1001 VVPI-1001 Basic Nucleofector Kit for Primary Mammalian Endothelial Cells 100 μl aluminum cuvette 4 × 25 reactionsEndothelial Cells and Media 37–41AExample for Nucleofection of primary porcine endothelial cells. Primaryporcine trabecular meshwork cells (derived from eye) were transfected byNucleofection <strong>with</strong> a plasmid encoding the green fluorescent maxGFPReporter Protein. 24 hours post Nucleofection, the cells were analyzed bylight (A) and fluorescence microscopy (B). (Data courtesy of Dr. Ted Acott,Oregon Health & Science University, USA.)BPage4Transfection / Nucleofector Kits for Primary Endothelial CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com205
Nucleofector Kits for Human Bronchial Epithelial Cells (NHBE)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of NHBEs usingthe different Nucleofection Platforms.ABOptimal kits for transfection of NHBEs in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. NHBE specifickits are available for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 65%––Viability: up to 53%Example of Nucleofection of NHBE. Clonetics Normal Human BronchialEpithelial Cells were transfected <strong>with</strong> pmaxGFP Vector. 24 hours postNucleofection, cells were analyzed by light (A) or fluorescence (B)microscopy.4Transfection / Nucleofector Kits for Primary Epithelial Cells■■Applications––Validated to work <strong>with</strong> Clonetics NHBE––Also tested <strong>with</strong> asthmatic and COPD bronchial epithelialcells––Easily verify previous cell line results in the analogousprimary cell typeOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPK-1001 VAPK-1001 Human Bronchial Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPK-1001 VPK-1001 Human Bronchial Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPK-1001 VVPK-1001 Human Bronchial Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageNHBE – Bronchial /Tracheal Epithelial Cells 52BEGM – Bronchial Epithelial Growth Medium BulletKit 52DHBE-As Diseased (Asthma) Bronchial/Tracheal Epithelial Cells 51DHBE-COPD Diseased (COPD) Bronchial/Tracheal Epithelial Cells 51206North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Mammary Epithelial CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of HMECs usingthe different Nucleofection Platforms.Optimal kits for transfection of HMECs in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. HMEC specifickits are available for the Nucleofector II/2b Device.AB■■Benefits––Transfection efficiency: up to 73%––Viability: up to 95%Example of Nucleofection of HMEC. Clonetics Human Mammary EpithelialCells were transfected <strong>with</strong> pmaxGFP Vector. 24 hours post Nucleofection,cells were analyzed by light (A) or fluorescence (B) microscopy.■■Applications––Validated to work <strong>with</strong> Clonetics HMEC––Easily verify previous cell line results in the analogousprimary cell type4Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPK-1002 VAPK-1002 Human Mammary Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPK-1002 VPK-1002 Human Mammary Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPK-1002 VVPK-1002 Human Mammary Epithelial Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageHMEC - Human Mammary Epithelial Cells 44MEGM - Mammary Epithelial Cell Growth Medium BulletKit 44Transfection / Nucleofector Kits for Primary Epithelial CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com207
Nucleofector Kits for Mammalian Epithelial Cells4Transfection / Nucleofector Kits for Primary Epithelial CellsFor mammalian epithelial cells lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The P1 and P3 Primary Cell Kits together <strong>with</strong> the cell-groupspecific Basic Protocols are suited for optimizations ofmammalian epithelial cells on the 4D-Nucleofector System,the 96-well Shuttle System or the 384-well NucleofectorSystem.A cell group-specific Basic Kit is suited for optimization ofmammalian epithelial cells using the Nucleofector II/2bDevice.■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols provide guidance through theoptimization procedure––Fine tuning of results is possible <strong>with</strong> help from ourScientific Support Team––Transfection efficiency: up to 83%––Viability: up to 98%Example for Nucleofection of primary renal proximal tubular epithelialcells. Human renal proximal tubular epithelial cells were transfected byNucleofection <strong>with</strong> a plasmid encoding the green fluorescent protein,eGFP. 48 hours post Nucleofection, cells were analyzed by fluorescencemicroscopy. (Data courtesy of C. Xu, R. L. Bacallao*, and S. L. Alper.Department of Medicine, Beth Israel Deaconess Medical Center andHarvard Medical School, Boston, USA, *University of Indiana School ofMedicine, Indianapolis, USA.)■■Aplications––Kits suited for epithelial cells from different mammalianspecies and various organs––Already tested for renal proximal tubular epithelial cells(RPTEC), Clonetics Epithelial Cells: human prostateepithelial cells (hPrEC) and human small airwayepithelial cells (SAEC)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-1012 V4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-1024 V4XP-1024 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-1032 V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-1096 V4SP-1096 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-1960 V4SP-1960 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-1002 V5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-1010 V5SP-1010 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1005 VAPI-1005 Basic Nucleofector Kit for Primary Mammalian Epithelial Cells 100 μl aluminum cuvette 10 reactionsVPI-1005 VPI-1005 Basic Nucleofector Kit for Primary Mammalian Epithelial Cells 100 μl aluminum cuvette 25 reactionsVVPI-1005 VVPI-1005 Basic Nucleofector Kit for Primary Mammalian Epithelial Cells 100 μl aluminum cuvette 4 × 25 reactions208North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Dermal Fibroblasts (NHDF)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of NHDF cellsusing the different Nucleofection Platforms.Optimal kits for transfection of NHDF cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P2 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. NHDF cellspecific kits are available for the Nucleofector II/2b Device.AB■■Benefits––Transfection efficiency: up to 90%––Viability: up to 98%––More than 90 publications citing Nucleofection ofhuman dermal fibroblasts■■Applications––Validated to work <strong>with</strong> Clonetics NHDF, neonatal andadult––Ideal for studying fibrosarcoma, fibrosis, scleroderma,or xeroderma pigmentosum––Optimal for both DNA and siRNA transfectionRelated ProductsNucleofection of adult human dermal fibroblasts <strong>with</strong> eGFP cDNA.Clonetics NHDF-Adult were transfected by Nucleofection using a plasmidencoding eGFP. 24 hours post Nucleofection, cells were analyzed by light(A) and fluorescence microscopy (B).www.lonza.com /citationsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-2012 V4XP-2012 P2 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-2024 V4XP-2024 P2 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-2032 V4XP-2032 P2 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-2096 V4SP-2096 P2 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-2960 V4SP-2960 P2 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-2002 V5SP-2002 P2 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-2010 V5SP-2010 P2 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPD-1001 VAPD-1001 Human Dermal Fibroblast Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPD-1001 VPD-1001 Human Dermal Fibroblast Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPD-1001 VVPD-1001 Human Dermal Fibroblast Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsNHDF-Ad – Human Adult Dermal Fibroblasts 35NHDF-Neo – Neonatal Human Dermal Fibroblasts 35FGM-2 – Fibroblast Growth Media BulletKit 36Page4Transfection / Nucleofector Kits for Primary FibroblastsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com209
Nucleofector Kits for Mouse Embryonic Fibroblasts (MEF)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of MEFs usingthe different Nucleofection Platforms.For the transfection of MEFs in the 4D-Nucleofector,96-well Shuttle or 384-well Nucleofector System werecommend using the Primary Cell Optimization Kits andthe respective optimization protocols. OptimalNucleofection Conditions are transferable between thesethree systems. MEF specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 62%––Viability: up to 88%Example of Nucleofection of primary MEFs. Spontaneously immortalizedmouse embryonic fibroblasts (strain: C57BL/6 × 129Sv) were transfectedby Nucleofection using a plasmid encoding the enhanced greenfluorescent protein eGFP. 24 hours post Nucleofection, the cells wereanalyzed by fluorescence microscopy. (Photograph courtesy of Dr. H.Hermanns and Prof. P.H. Heinrich, University of Aachen, Germany.)4■■Applications––Kits suitable for various mouse embryonic fibroblastclonesTransfection / Nucleofector Kits for Primary FibroblastsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVPD-1006 VPD-1006 Mouse Embryonic Fibroblast Starter Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVAPD-1004 VAPD-1004 Mouse Embryonic Fibroblast Nucleofector Kit 1 100 μl aluminum cuvette 10 reactionsVAPD-1005 VAPD-1005 Mouse Embryonic Fibroblast Nucleofector Kit 2 100 μl aluminum cuvette 10 reactionsVPD-1004 VPD-1004 Mouse Embryonic Fibroblast Nucleofector Kit 1 100 μl aluminum cuvette 25 reactionsVPD-1005 VPD-1005 Mouse Embryonic Fibroblast Nucleofector Kit 2 100 μl aluminum cuvette 25 reactionsVVPD-1004 VVPD-1004 Mouse Embryonic Fibroblast Nucleofector Kit 1 100 μl aluminum cuvette 4 × 25 reactionsVVPD-1005 VVPD-1005 Mouse Embryonic Fibroblast Nucleofector Kit 2 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsMouse Embryonic Fibroblasts (as feeder cells) 67Dulbecco’s Modified Eagle’s Medium 103Page210North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mammalian FibroblastsFor mammalian fibroblasts lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The P2 and P3 Primary Cell Kits are suited for optimizationsof mammalian fibroblasts on the 4D-Nucleofector System,the 96-well Shuttle System or the 384-well NucleofectorSystem.A cell group-specific Basic Kit is suited for optimization ofmammalian fibroblasts using the Nucleofector II/2bDevice.■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols guiding through the optimizationprocedure––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team––Transfection efficiency: up to 90%––Viability: up to 98%■■Applications––Kits suited for fibroblasts from different mammalianspecies and various organs––Already tested for macaque dermal fibroblasts, bovinefibroblasts, human colon myofibroblasts, mouse lungfibroblasts, etc.4Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-2012 V4XP-2012 P2 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-2024 V4XP-2024 P2 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-2032 V4XP-2032 P2 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-2096 V4SP-2096 P2 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-2960 V4SP-2960 P2 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-2002 V5SP-2002 P2 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-2010 V5SP-2010 P2 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1002 VAPI-1002 Basic Nucleofector Kit for Primary Mammalian Fibroblasts 100 μl aluminum cuvette 10 reactionsVPI-1002 VPI-1002 Basic Nucleofector Kit for Primary Mammalian Fibroblasts 100 μl aluminum cuvette 25 reactionsVVPI-1002 VVPI-1002 Basic Nucleofector Kit for Primary Mammalian Fibroblasts 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsHuman Fibroblast Cells 34-36PageTransfection / Nucleofector Kits for Primary FibroblastsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com211
Nucleofector Kits for Human HepatocytesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanhepatocytes using the different Nucleofection Platforms.Optimal kits for transfection of human hepatocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols.AB■■Benefits––Transfection efficiency: up to 54%––Viability: up to 69%––Cells retain their functionality for up to 120 hours––Efficient non-viral transfection of non or low proliferatingcellsExample showing typical Nucleofection results of human hepatocytes.Cryopreserved human hepatocytes were transfected <strong>with</strong> pmaxGFPVector. 120 hours post Nucleofection, cells were analyzed by light (A) andfluorescence microscopy (B).4Transfection / Nucleofector Kits for Primary Hepatocytes■■Applications––Excellent transfection rates for DNA and siRNA––Study metabolic pathways and toxic effects of newtherapeutic agentsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Related ProductsHCM Hepatocyte Culture Media BulletKit 49Page212North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mouse or Rat HepatocytesVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mouse or rathepatocytes using the different Nucleofection Platforms.For the transfection of mouse or rat hepatocytes in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System we recommend using the PrimaryCell Optimization Kits and the respective optimizationprotocols. Optimal Nucleofection Conditions aretransferable between these three systems. Mouse andrat hepatocyte specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 54%––Viability: up to 80%––Cells retain functional properties■■Applications––Kits suitable for mouse or rat hepatocytes––Suited for DNA and siRNA transfections––Ideal for research on new therapeutic agents andtoxicity mechanismsRelated ProductsN = nuclei; * = bile canaliculiHepatocytes transfected by Nucleofection maintain their morphologyand polarization. Primary rat hepatocytes were transfected byNucleofection <strong>with</strong> the pmaxGFP Vector (A) or a plasmid containing thecDNA sequence for a plasma membrane receptor-YFP fusion protein (B).Cells were stained <strong>with</strong> antibodies against desmoplakin (A; blue) tovisualize cell boundaries and against multidrug resistance protein 2(MRP2; A+B; red) to show the apical, canalicular membrane. maxGFPReporter Protein was located in the cytosol of transfected cells (A).YFP-fusion protein was correctly targeted to both the basolateral and theapical membrane domain as shown by co-localization <strong>with</strong> MRP2 (B).These data prove normal formation of bile canaliculi in hepatocytestransfected by Nucleofection. (Data courtesy of V. Keitel, F. Schliess and D.Häussinger, Department for Gastroenterology, Hepatology andInfectiology, Heinrich-Heine-University Düsseldorf, Germany.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPL-1004 VAPL-1004 Mouse/Rat Hepatocyte Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPL-1004 VPL-1004 Mouse/Rat Hepatocyte Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPL-1004 VVPL-1004 Mouse/Rat Hepatocyte Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsHCM Hepatocyte Culture Media BulletKit 49ANN* * NNNB**Page4Transfection / Nucleofector Kits for Primary HepatocytesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com213
Nucleofector Kits for Human Aortic Smooth Muscle Cells (AoSMC)4Transfection / Nucleofector Kits for Primary Muscle CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of humanAoSMCs using the different Nucleofection Platforms.Optimal kits for transfection of human AoSMCs in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P1 Primary Cell Kits used incombination <strong>with</strong> respective basic protocols for mammaliansmooth muscle cells. Human AoSMC specific kits areavailable for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 80%––Viability: up to 96%––10-fold higher transfection efficiency compared tolipofection■■Applications––Kit suitable for human aortic and vascular smoothmuscle cells––Applicable for transient long-term expression up tothree weeks––Ideal tool for studies on human vascular disorders, suchas atherosclerosis and stroke% Transfected cells10090807060504030201001 day 2 days 7 days 14 days 21 days 28 daysTime course of transient expression of transfected human AoSMC.Clonetics Human AoSMC were transfected by Nucleofection using aplasmid encoding the mouse MHC class I heavy chain molecule H-2Kk. 1,2, 7, 14, 21, and 28 days post Nucleofection, the cells were analyzed fortheir H-2Kk expression by flow cytometry. Dead cells were excluded fromthe analysis by propidium iodide staining and gating.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-1012 V4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-1024 V4XP-1024 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-1032 V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-1096 V4SP-1096 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-1960 V4SP-1960 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-1002 V5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-1010 V5SP-1010 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPC-1001 VAPC-1001 Human Aortic Smooth Muscle Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPC-1001 VPC-1001 Human Aortic Smooth Muscle Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPC-1001 VVPC-1001 Human Aortic Smooth Muscle Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageAoSMC – Human Aortic Smooth Muscle Cells 32SmGM-2 Smooth Muscle Cell Growth Media BulletKit 30,33214North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Skeletal Muscle MyoblastsOptimal kits for transfection of human skeletal muscle cells(HSMM) in the 4D-Nucleofector X Unit are the P5 PrimaryCell Kits, used in combination <strong>with</strong> the cell-type specificprotocol. Due to transferability between all platforms, sameconditions apply for the 96-well Shuttle or 384-wellNucleofector Systems.A■■Benefits––Transfection efficiency: up to 78%––Viability: up to 62%■■Applications––Validated to work <strong>with</strong> Clonetics HSMM––Easily verify previous cell line results in the analogousprimary cell typeB4Example of Nucleofection of HSMM. Clonetics Human Skeletal MuscleMyoblasts were transfected <strong>with</strong> pmaxGFP Vector. 24 hours postNucleofection, cells were analyzed by light (A) or fluorescence (B)microscopy.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-5012 V4XP-5012 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-5024 V4XP-5024 P5 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-5032 V4XP-5032 P5 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-5096 V4SP-5096 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-5960 V4SP-5960 P5 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-5002 V5SP-5002 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-5010 V5SP-5010 P5 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Related ProductsPageHSMM – Human Skeletal Muscle Myoblasts 61SkGM – Skeletal Muscle Cell Growth Media BulletKit 61Transfection / Nucleofector Kits for Primary Muscle CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com215
Nucleofector Kits for Mammalian Smooth Muscle Cells4Transfection / Nucleofector Kits for Primary Muscle CellsFor mammalian smooth muscle cells lacking a cell-typespecific Optimized Protocol, we offer a selection of kits thatcan be used to easily define optimal NucleofectionConditions.The P1 Primary Cell Kits together <strong>with</strong> the cell-group specificBasic Protocols are suited for optimizations of mammaliansmooth muscle cells on the 4D-Nucleofector System, the96-well Shuttle System or the 384-well NucleofectorSystem.A cell-group specific Basic Kit is suited for optimization ofmammalian smooth muscle cells using the NucleofectorII/2b Device.■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols guiding through the optimizationprocedure––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team––Transfection efficiency: up to 95%––Viability: up to 96%ANucleofection of primary smooth muscle cells. Primary pulmonary arterysmooth muscle cells were transfected <strong>with</strong> pmaxGFP Vector. 24 hourspost Nucleofection, cells were analyzed by light (A) and fluorescence (B)microscopy.■■Applications––Kits suited for smooth muscle cells from differentmammalian species and various organs––Already tested for porcine vascular smooth musclecells and coronary artery smooth muscle cells(Clonetics CASMC)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-1012 V4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-1024 V4XP-1024 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-1032 V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-1096 V4SP-1096 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-1960 V4SP-1960 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-1002 V5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-1010 V5SP-1010 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1004 VAPI-1004 Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells 100 μl aluminum cuvette 10 reactionsVPI-1004 VPI-1004 Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells 100 μl aluminum cuvette 25 reactionsVVPI-1004 VVPI-1004 Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageHuman Smooth Muscle Cells 32SmGM-2 Smooth Muscle Cell Growth Media BulletKit 30,33B216North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Chicken NeuronsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of chickenneurons using the different Nucleofection Platforms.Optimal kits for transfection of chicken neurons in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> respective basic protocols for mammalianneurons. Chicken neuron specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 43%––Transgene expression for more than one week––Cells retain morphological and functional propertiesACB■■Applications––Kits suitable for hippocampal neurons and dorsal rootganglia neurons4Nucleofection in adherent state is possible using theAD1 4D-Nucleofector Y Kitwww.lonza.com/celldatabaseFormation of normal growth cones indicates maintenance of functionalityof dorsal root ganglia after Nucleofection. DRG neurons from chickenwere transfected by Nucleofection <strong>with</strong> a plasmid encoding the GFPprotein. After cultivation on pre-coated glass coverslips overnight, singlecells were analyzed for formation of normal growth cones (A – C), F-actinlocalization after staining <strong>with</strong> Alexa 568 conjugated phalloidin (A and C)and GFP expression (B and C). (Photograph courtesy of B. Eickholt, King’sCollege, London, Great Britain.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPG-1002 VAPG-1002 Chicken Neuron Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPG-1002 VPG-1002 Chicken Neuron Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPG-1002 VVPG-1002 Chicken Neuron Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsTransfection / Nucleofector Kits for Primary Neural CellsRelated ProductsPagePNGM Primary Neuron Growth Media BulletKit 70-71Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com217
Nucleofector Kits for Mouse Neurons4Transfection / Nucleofector Kits for Primary Neural CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mouseneurons using the different Nucleofection Platforms.Optimal kits for transfection of mouse neurons in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> respective basic protocols for mammalianneurons. Mouse neuron specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 60%––Viability: up to 65 %––Transgene expression for more than one week––Cells retain morphological and functional properties■■Applications––Kits suitable for hippocampal, cortical and dorsal rootganglia neuronsNucleofection in adherent state is possible using theAD1 4D-Nucleofector Y Kit––More than 50 peer-reviewed publicationsRelated ProductsANucleofection of primary mouse hippocampal neurons. Primarydissociated neurons of mixed glial cultures were transfected using aplasmid encoding the enhanced green fluorescent protein eGFP. 48 hourspost Nucleofection, the cells were analyzed by light (A) and fluorescencemicroscopy (B). (Photograph courtesy of A. Dityatev, G. Dityateva and M.Hammond, Center for Molecular Neurobiology, Hamburg, Germany.)www.lonza.com/celldatabasewww.lonza.com/citationsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPG-1001 VAPG-1001 Mouse Neuron Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPG-1001 VPG-1001 Mouse Neuron Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPG-1001 VVPG-1001 Mouse Neuron Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsPrimary Mouse Neural Cells 70PNGM Primary Neuron Growth Media BulletKit 70-71BPage218North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Rat NeuronsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of rat neuronsusing the different Nucleofection Platforms.Optimal kits for transfection of rat neurons in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Rat neuronspecific kits are available for the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 67% using a non-viralmethod––Viability: up to 60%––Cells retain morphological and functional properties––Transgene expression for more than one week■■Applications––Kits suitable for hippocampal neurons, cortical neuronsand dorsal root ganglia neurons––Proven performance for siRNA, shRNA, miRNA, andantisense oligonucleotidesNucleofection in adherent state is possible using theAD1 4D-Nucleofector Y Kit––Reaching 250 peer-reviewed publicationsRelated ProductsCryopreserved dissociated rat DRG cells were thawed and cultured in24-well plates for Nucleofection using 4D-Nucleofector Y Unit. DRG cellculture was transfected at 2 DIV and fixed 24 hours post Nucleofection(program EH-166). Neuronal networks are stained using anti Tuj-1antibody (red; personal gift W. Staines). Transfected neurons and Schwanncells can be seen in green (maxGFP Protein).www.lonza.com/celldatabasewww.lonza.com/citationsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPG-1003 VAPG-1003 Rat Neuron Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPG-1003 VPG-1003 Rat Neuron Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPG-1003 VVPG-1003 Rat Neuron Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsClonetics Primary Rat Neural Cells 70PNGM Primary Neuron Growth Media BulletKit 70-71Page4Transfection / Nucleofector Kits for Primary Neural CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com219
Nucleofector Kits for Mammalian NeuronsFor mammalian neurons lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The P3 Primary Cell Kits together <strong>with</strong> the cell-group specificBasic Protocols are suited for optimizations of mammalianneurons on the 4D-Nucleofector System, the 96-wellShuttle system or the 384-well Nucleofector System.A cell-group specific Basic Kit is suited for optimization ofmammalian neurons using the Nucleofector II/2b Device.% Transfection efficiency706050403020100ElectroporationDsRedLipofectionEGFPNucleofection4■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols provide guidance through theoptimization procedure––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team––Transfection efficiency: up to 92%––Viability: up to 80%Comparison of conventional electroporation, lipofection andNucleofection for transfection of rat neuronal progenitor cells. Ventralmesencephalic progenitor (VMP) cells from rat brain, which are theimportant source of dopaminergic neurons for cell replacement strategiesin Parkinson’s disease, were transfected <strong>with</strong> two different plasmidsexpressing DsRed or eGFP. For transfection, conventional electroporation(EasyjecT from EquiBio, 100 µg plasmid per 500,000 cells), lipofection(Lipofectamine 2000 Reagent, 0.5 µg DNA per 60,000 cells), orNucleofection (5 µg DNA per 2,000,000 cells) were used. (Data fromCesnulevicius et al. (2006) Stem Cells 24(12), 2776-91.)Transfection / Nucleofector Kits for Primary Neural Cells■■Applications––Kits suited for various neuron types from differentmammalian speciesNucleofection in adherent state is possible using theAD1 4D-Nucleofector Y KitOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1003 VAPI-1003 Basic Nucleofector Kit for Primary Mammalian Neurons 100 μl aluminum cuvette 10 reactionsVPI-1003 VPI-1003 Basic Nucleofector Kit for Primary Mammalian Neurons 100 μl aluminum cuvette 25 reactionsVVPI-1003 VVPI-1003 Basic Nucleofector Kit for Primary Mammalian Neurons 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPNGM Primary Neural Cell Growth Media BulletKit 70-71Page220North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mammalian Glial CellsA selection of kits for mammalian glial cells helps you toeasily define optimal Nucleofection Conditions.The P3 Primary Cell Kits together <strong>with</strong> the cell-group specificBasic Protocols are suited for optimizations of mammalianglial cells on the 4D-Nucleofector System, the 96-wellShuttle System or the 384-well Nucleofector System.A cell-group specific Basic Kit is suited for optimization ofmammalian glial cells using the Nucleofector II/2b Device.AB■■Benefits––Optimizations can be performed <strong>with</strong>in one experiment––Detailed protocols guide through optimization procedure––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team––Transfection efficiency: up to 67%––Viability: up to 80%CD4■■Applications––Kits suited for various glial cells from differentmammalian species––Already tested for rat and mouse astrocytes, ratoligodendrocytesNucleofection in adherent state using the AD14D-Nucleofector Y KitExpression of GFAP and eGFP in rat astrocytes transfected byNucleofection. Primary rat astrocytes were isolated from rat embryos(E17) and cultured for 10 days until cells reached confluency. These cellswere transfected <strong>with</strong> a plasmid encoding the enhanced green fluorescentprotein, eGFP. 24 hours post Nucleofection, cells were analyzed byfluorescence microscopy for expression of eGFP (A) and GFAP (B), anastrocyte-specific marker protein. Nuclei were stained <strong>with</strong> DAPI (C). (D)shows an overlay of the images. (Photographs courtesy of Dr. Hyun-JuKim and Dr. Tim Vartanian, Beth Israel Deaconess Medical Center, Dept. ofNeurology, Boston, Massachusetts, USA.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4YP-1A24 V4YP-1A24 AD1 4D-Nucleofector Y Kit 24-well Dipping Electrode 24 reactions96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPI-1006 VAPI-1006 Basic Nucleofector Kit for Primary Mammalian Glial Cells 100 μl aluminum cuvette 10 reactionsVPI-1006 VPI-1006 Basic Nucleofector Kit for Primary Mammalian Glial Cells 100 μl aluminum cuvette 25 reactionsVVPI-1006 VVPI-1006 Basic Nucleofector Kit for Primary Mammalian Glial Cells 100 μl aluminum cuvette 4 × 25 reactionsTransfection / Nucleofector Kits for Primary Neural CellsRelated ProductsPagePrimary Neural Cells and Media 70Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com221
Nucleofector Kits for Human CD34 + CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of human CD34 +cells using the different Nucleofection Platforms.Optimal kits for transfection of human CD34 + cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Human CD34 +cell specific kits are available for the Nucleofector II/2bDevice.% Positive cells50403020104Transfection / Nucleofector Kits for Primary Stem Cells■■Benefits––Transfection efficiency: up to 83%––Viability: up to 70%––No influence on hematopoietic cell differentiation––Both fresh or cryopreserved material can be used■■Applications––Kits suitable for unstimulated human CD34 + bonemarrow cells––Cells can be derived from cord blood or leukapheresismaterial––Cited for DNA and siRNA transfection043684120200HoursPeripheral blood stem cellsBone marrow stem cellsLong-term transgene expression after Nucleofection of blood and bonemarrow derived CD34 + cells. Kinetics of deltaLNGFR (Low Affinity NerveGrowth Factor Receptor) expression were determined by flow cytometricanalysis. 39 ± 5.9% of peripheral blood stem cells showed deltaLNGFRstaining 4 hours after Nucleofection <strong>with</strong> a continuous decrease (n = 3, 3patients). Bone marrow stem cells showed maximal deltaLNGFRexpression <strong>with</strong> 26 ± 9.7% 84 hours after transfection, which thendecreased in the proliferating culture (n = 3, single patient). (Datacourtesy of Greiner et al., University Hospital of Ulm, Ulm, Germany.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPA-1003 VAPA-1003 Human CD34 + Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPA-1003 VPA-1003 Human CD34 + Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPA-1003 VVPA-1003 Human CD34 + Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageHuman CD34 + Progenitor Cells 80RPMI 1640 <strong>with</strong>out L-glutamine 110X-VIVO 15 Chemically Defined, Serum-free Hematopoietic Cell Medium 121HPGM Hematopoietic Progenitor Growth Medium 81222North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human H9 Stem CellsVarious Nucleofector Kits and correspondingOptimized Protocols are available for the transfection ofhuman H9 stem cells using the different NucleofectionPlatforms.Optimal kits for transfection of human H9 stem cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. A stem cellspecific Basic Kit is suited for optimization of human stemcells using the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 64%––Viability: up to 98%––Excellent preservation of pluripotency■■Applications––Kits suitable for human H9 stem cells––Elucidate various aspects of stem cell differentiationH9 cells preserve pluripotency post Nucleofection. H9 cells transfected<strong>with</strong> the pmaxGFP Vector maintain their undifferentiated state. Analysisafter 24 hours shows expression of maxGFP Protein (green) as well as ofthe pluripotency markers SSEA4 (red) and Oct4 (purple). The blue signalsrefer to nuclear staining by DAPI. (Data kindly provided by Jennifer Moore,Rutgers University, Piscataway, USA.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVPH-5002 VPH-5002 Human Stem Cell Nucleofector Starter Kit 100 μl aluminum cuvette 18 reactionsVAPH-5012 VAPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 10 reactionsVPH-5012 VPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 25 reactionsVVPH-5012 VVPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 4 × 25 reactionsVAPH-5022 VAPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 10 reactionsVPH-5022 VPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 25 reactionsVVPH-5022 VVPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 4 × 25 reactions4Transfection / Nucleofector Kits for Primary Stem CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com223
Nucleofector Kits for Human Mesenchymal Stem Cells (MSC)4Transfection / Nucleofector Kits for Primary Stem CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of human MSCusing the different Nucleofection Platforms.Optimal kits for transfection of human MSC in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P1 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Human MSCcell specific kits are available for the Nucleofector II/2bDevice.■■Benefits––Transfection efficiency: up to 88%––Viability: up to 86%––Maintenance of functional properties––Efficient non-viral transfection of human MSC■■Applications––Validated to work <strong>with</strong> Poietics MSC––Differentiation of transfected MSC into adipocytes orosteoblastsRelated Products% Transfection efficiency/viability1101009080706050403020100NucleofectionReagent FDOTAPTransfection efficiencyViabilityComparison of Nucleofection <strong>with</strong> lipofection for transfection of humanMSC. MSC were transfected by Nucleofection <strong>with</strong> pcDNA3/NT-GFP usingeither Nucleofection or the lipid-based Fugene® 6 or DOTAP Reagents (bothRoche Applied Science). MSC transfected by Nucleofection were analyzedfor transfection efficiency roughly 60 hours post Nucleofection, cellstransfected <strong>with</strong> Fugene® 6 or DOTAP Reagents were analyzed after 72hours. Transfection efficiency was scored by flow cytometric analysis andreported as percentage of GFP+ cells. The percentage of viable cells wasestimated by trypan blue exclusion. (Data courtesy of Aluigi M, Fogli M,Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D’Errico-Grigioni A, Ferri E, Baccarani M and Lemoli RM, Institute of Hematology andMedical Oncology, Bologne, Italy).Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-1012 V4XP-1012 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-1024 V4XP-1024 P1 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-1032 V4XP-1032 P1 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-1096 V4SP-1096 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-1960 V4SP-1960 P1 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-1002 V5SP-1002 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-1010 V5SP-1010 P1 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPE-1001 VAPE-1001 Human Mesenchymal Stem Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPE-1001 VPE-1001 Human Mesenchymal Stem Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPE-1001 VVPE-1001 Human Mesenchymal Stem Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionshMSC Human Mesenchymal Stem Cells 87MSCGM Mesenchymal Stem Cell Growth Medium BulletKit 88hMSC Mesenchymal Stem Cell Adipogenic Differentiation BulletKit 88hMSC Mesenchymal Stem Cell Osteogenic Differentiation BulletKit 87hMSC Mesenchymal Stem Cell Chondrogenic Differentiation BulletKit 87Trypsin/EDTA for Mesenchymal Stem Cells 88Page224North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Human Stem CellsFor human stem cells lacking a cell-type specific OptimizedProtocol, we offer a selection of kits that can be used toeasily define optimal Nucleofection Conditions.The P3 and P4 Primary Cell Kits together <strong>with</strong> the cell- groupspecific Basic Protocols are suited for optimizations ofhuman stem cells on the 4D-Nucleofector System, the96-well Shuttle System or the 384-well NucleofectorSystem.A cell-group specific Basic Kit is suited for optimization ofhuman stem cells using the Nucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 95%––Viability: up to 98%––Circumvents tedious creation of viruses––Less DNA and lower cell number required% Transfection efficiency / viability1009080706050403020100H1H9H9.2Hues-9BGO1VCustomerTransfection EfficiencyViabilityTransfection efficiencies of human stem cell lines. Different human stemcell lines were transfected by Nucleofection using the pmaxGFP Vector.(Data for Nucleofection of human stem cells are compiled fromexperiments performed by leading stem cell research customers.)4■■Applications––Already tested for H1, H9, H14 and HS306 cellsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4XP-4012 V4XP-4012 P4 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-4024 V4XP-4024 P4 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-4032 V4XP-4032 P4 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SP-4096 V4SP-4096 P4 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-4960 V4SP-4960 P4 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SP-4002 V5SP-4002 P4 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-4010 V5SP-4010 P4 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVPH-5002 VPH-5002 Human Stem Cell Nucleofector Starter Kit 100 μl aluminum cuvette 18 reactionsVAPH-5012 VAPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 10 reactionsVPH-5012 VPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 25 reactionsVVPH-5012 VVPH-5012 Human Stem Cell Nucleofector Kit 1 100 μl aluminum cuvette 4 × 25 reactionsVAPH-5022 VAPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 10 reactionsVPH-5022 VPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 25 reactionsVVPH-5022 VVPH-5022 Human Stem Cell Nucleofector Kit 2 100 μl aluminum cuvette 4 × 25 reactionsTransfection / Nucleofector Kits for Primary Stem CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com225
Nucleofector Kits for Mouse Embryonic Stem (ES) CellsVarious Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mouse EScells using the different Nucleofection Platforms.Optimal kits for transfection of mouse ES cells in the4D-Nucleofector, 96-well Shuttle or 384-wellNucleofector System are the P3 Primary Cell Kits used incombination <strong>with</strong> cell-type specific protocols. Mouse ES cellspecific kits are available for the Nucleofector II/2b Device.NucleofectionElectroporation4Transfection / Nucleofector Kits for Primary Stem Cells■■Benefits––Transfection efficiency: up to 90%––Viability: up to 99%––Homogenous transient gene expression pattern––Preservation of cell functionality (ability to differentiate)■■Applications––Tested <strong>with</strong> several mouse ES cell lines (e.g., R1, D3,E14)––Successfully used to generate germline chimerasno DNAComparison of Nucleofection and electroporation for transfection ofmouse ES cells. Mouse ES cells were transfected by Nucleofection andcompared to mock-transfected (no DNA) and electroporated ES cells usingBio-Rad® Gene Pulser®. Cells were stained 48 hours after transfection fortransient lacZ expression. (Data courtesy of S. Boljahn, A. Rode, M. Joao daSilva, T. Hennek and B. Zevnik, Artemis Pharmaceutical GmbH, Cologne,Germany.)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-3012 V4XP-3012 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XP-3024 V4XP-3024 P3 Primary Cell 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XP-3032 V4XP-3032 P3 Primary Cell 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SP-3096 V4SP-3096 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SP-3960 V4SP-3960 P3 Primary Cell 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)384-well Nucleofector KitsV5SP-3002 V5SP-3002 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SP-3010 V5SP-3010 P3 Primary Cell 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Nucleofector II/2b KitsVAPH-1001 VAPH-1001 Mouse Embryonic Stem Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPH-1001 VPH-1001 Mouse Embryonic Stem Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPH-1001 VVPH-1001 Mouse Embryonic Stem Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsRelated ProductsPageMouse Embryonic Fibroblasts (as feeder cells) 67DMEM 4.5 g/L glucose <strong>with</strong> L-glutamine 103226North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Mouse Neural Stem Cells (NSC)Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of mouse NSCusing the different Nucleofection Platforms.ABFor the transfection of mouse NSC in the 4D-Nucleofector,96-well Shuttle or 384-well Nucleofector System werecommend using the Primary Cell Optimization Kits andthe respective optimization protocols. OptimalNucleofection Conditions are transferable between thesethree systems. Mouse NSC specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 82%––Viability: up to 90%––Transgene expression for several daysNucleofection of mouse NSCs. Primary NSCs isolated from the lateralventrical wall of an adult mouse were transfected by Nucleofection usinga plasmid encoding the enhanced green fluorescent protein eGFP. 48hours post Nucleofection, the cells were analyzed by light (A) andfluorescence microscopy (B). (Photograph courtesy of Dr. L. Wikstrom etal., NeuroNova, Stockholm, Sweden.)4■■Applications––Kits suitable for mouse neurospheres and adherentcells––Differentiation into neurons and astrocytes possibleOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPG-1004 VAPG-1004 Mouse Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPG-1004 VPG-1004 Mouse Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPG-1004 VVPG-1004 Mouse Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactionsTransfection / Nucleofector Kits for Primary Stem CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com227
Nucleofector Kits for Rat Neural Stem Cells (NSC)4Various Nucleofector Kits and corresponding OptimizedProtocols are available for the transfection of rat NSC usingthe different Nucleofection Platforms.For the transfection of rat NSC in the 4D-Nucleofector,96-well Shuttle or 384-well Nucleofector System werecommend using the Primary Cell Optimization Kits andthe respective optimization protocols. OptimalNucleofection Conditions are transferable between thesethree systems. Rat NSC specific kits are available for theNucleofector II/2b Device.■■Benefits––Transfection efficiency: up to 46%––Efficient non-viral method for efficient gene transferinto primary neural stem cells––Transgene expression for several daysABNucleofection of rat NSC. Primary NSC isolated from rat embryos (E14)were transfected by Nucleofection using a plasmid encoding enhancedgreen fluorescent protein eGFP under control of an EF1alpha promoter(pcDNAEF1-eGFP). Post Nucleofection, cells were cultured <strong>with</strong> bFGF for 2days, then for 5 additional days <strong>with</strong>out bFGF to differentiate into neurons.Cells were analyzed 2 days (A) and 7 days (B) post Nucleofection byfluorescence microscopy. (Photograph courtesy of S.H. Lee, College ofMedicine, Dept. of Biochemistry, Hanyang University, Seoul, South Korea.)Transfection / Nucleofector Kits for Primary Stem Cells■■Applications––Kits suitable for rat neurospheres and adherent cells––Differentiation into neurons and astrocytes possibleOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Nucleofector II/2b KitsVAPG-1005 VAPG-1005 Rat Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 10 reactionsVPG-1005 VPG-1005 Rat Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 25 reactionsVVPG-1005 VVPG-1005 Rat Neural Stem Cell Nucleofector Kit 100 μl aluminum cuvette 4 × 25 reactions228North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kits for Animal Stem CellsFor animal stem cells cells lacking a cell-type specificOptimized Protocol, we offer a selection of kits that can beused to easily define optimal Nucleofection Conditions.The Primary Cell Optimization Kits are suited foroptimizations of stem cells on the 4D-Nucleofector System,the 96-well Shuttle System or the 384-well NucleofectorSystem.■■Benefits––Protocols provide guidance through the optimizationprocedure––Optimizations can be performed <strong>with</strong>in one experiment––Fine tuning of results is possible <strong>with</strong> the help of ourScientific Support Team■■Applications––Kits suited for stem cells from different mammalianspecies and various organs4Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size4D-Nucleofector KitsV4XP-9096 V4XP-9096 Primary Cell Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 96 reactions (16-well)96-well Shuttle KitsV4SP-9096 V4SP-9096 Primary Cell Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 160 reactions (96-well)384-well Nucleofector KitsV5SP-9001 V5SP-9001 Primary Cell Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Related ProductsPageNucleofector Kits for Human Stem Cells 223,225Transfection / Nucleofector Kits for Primary Stem CellsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com229
Cell Line Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systems4We offer three different Cell Line Nucleofector Solutions SE,SF and SG for the 4D-Nucleofector, 96-well Shuttle andthe 384-well Nucleofector Systems.Each Cell Line Kit Contains––Specific Nucleofector Solution SE, SF or SG––Supplement––pmaxGFP Control Vector––Either single 100 µl Nucleocuvettes, 16-wellNucleocuvette Strips, 96-well or 384-wellNucleocuvette PlatesAll kits are available in various sizes (please refer to orderinginformation for details). Optimized Protocols outlining theoptimal Nucleofector Kit for a broad selection of cell linesare available and can be downloaded from our website. Youcan always find the most up-to-date information in ouronline cell database.■■Benefits––Each of the three Nucleofector Solutions can be usedfor a selection of different cell lines––Conditions are transferable between 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector Systemsand between 20 and 100 µl Nucleocuvettes■■Applications––Transfection of lower cell numbers (from 2 × 10 4 to1 × 10 6 cells) to higher cell numbers (from 2 × 10 5 to2 × 10 7 cells) is possible––Flexible throughput from single cuvette (100 µl) to16-well Nucleocuvette Strip (20 µl), 96-well and384-well Nucleocuvette Plates is possiblewww.lonza.com/celldatabasewww.lonza.com/protocolsTransfection / Nucleofector Kits for Cell Lines100 µl Nucleocuvette(4D-Nucleofector System)16-well Nucleocuvette Strip(4D-Nucleofector System)96-well Nucleocuvette Plate(96-well Shuttle System)Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size384-well Nucleocuvette Plate(384-well Nucleofector System)4D-Nucleofector KitsV4XC-1012 V4XC-1012 SE Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XC-1024 V4XC-1024 SE Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XC-1032 V4XC-1032 SE Cell Line 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4XC-2012 V4XC-2012 SF Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XC-2024 V4XC-2024 SF Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XC-2032 V4XC-2032 SF Cell Line 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)V4XC-3012 V4XC-3012 SG Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 12 reactionsV4XC-3024 V4XC-3024 SG Cell Line 4D-Nucleofector X Kit L 100 μl Nucleocuvette 24 reactionsV4XC-3032 V4XC-3032 SG Cell Line 4D-Nucleofector X Kit S 20 μl Nucleocuvette 32 reactions (16-well)96-well Shuttle KitsV4SC-1096 V4SC-1096 SE Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SC-1960 V4SC-1960 SE Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SC-2096 V4SC-2096 SF Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SC-2960 V4SC-2960 SF Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)V4SC-3096 V4SC-3096 SG Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)V4SC-3960 V4SC-3960 SG Cell Line 96-well Nucleofector Kit 20 μl Nucleocuvette 960 reactions (96-well)230North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Line Kits for 4D-Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsContinuedOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size384-well Nucleofector KitsV5SC-1002 V5SC-1002 SE Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SC-1010 V5SC-1010 SE Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SC-2002 V5SC-2002 SF Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SC-2010 V5SC-2010 SF Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)V5SC-3002 V5SC-3002 SG Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 768 reactions (384-well)V5SC-3010 V5SC-3010 SG Cell Line 384-well Nucleofector Kit 20 μl Nucleocuvette 3840 reactions (384-well)Quick Reference Guide – Cell Line KitsCell line Efficiency Viable cells Solution 100 µl (12 rxn)Cat. No.Kits for 4D-Nucleofector (Cat. No.)100 µl (24 rxn)Cat. No.20 µl (32 rxn)Cat. No.Kits for 96-well Shuttle (Cat. No.)20 µl (96 rxn)Cat. No.20 µl (960 rxn)Cat. No.293 83% 93% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-29603T3-L1 pre-ad 97% 66–79% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960A20 80% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960A549 81% 62% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960ARPE-19 87% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960Ba / F3 80% 60–70% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960Beta TC-6 66–77% 49–82% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960BHK-21 97–98% 91–95% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960C6 92% 55–70% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960CHO-K1 86% 97% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960CHO-S [suspension] 86% 55–57% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960COS-7 91–99% 80–96% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960DU 145 89% 86–92% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960EL4 70–80% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960GH3 60–80% 60–70% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960H9C2 80–90% 54–72% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960HCT 116 70–80% 65–75% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960HeLa 75% 89% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960HeLa S3 61–85% 62–95% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960Hep G2 95.50% 92.70% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960HL-60 58% 61% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960HT29 51–67% 60% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960IMR32 74–86% 45–63% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960IMR90 65% 70% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960Jurkat 92% 71–80% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960K-562 92% 95% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960L-428 70–80% 85% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960LnCAP 70% 45% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960MCF7 72% 89% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960MDA-MB-231 73–89% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960MDCK 72–82% 50–55% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960MG63 70–73% 60–65% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960MRC-5 84–86% 67–73% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-19604Transfection / Nucleofector Kits for Cell LinesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com231
4Transfection / Nucleofector Kits for Cell LinesCell Line Kits for 4D Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsContinuedQuick Reference Guide – Cell Line KitsCell line Efficiency Viable cells Solution 100 µl (12 rxn)Cat. No.Kits for 4D-Nucleofector (Cat. No.)100 µl (24 rxn)Cat. No.20 µl (32 rxn)Cat. No.Kits for 96-well Shuttle (Cat. No.)20 µl (96 rxn)Cat. No.20 µl (960 rxn)Cat. No.Neuro-2a [N2a] 67% 82% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960NIH/3T3 95% 93% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960PC3 83% 79% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960Raji 65–69% 71% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960Ramos 40–51% 70–77% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960RAW 264.7 60% 86% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960RIN-m5F 68–90% 71–85% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960Sf9 100% 48–64% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960SH-SY5Y 81% 80% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960Sp2-0 65–69% 80–90% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960T-47D 80% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960T84 88% 50–70% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960THP-1 65% 81% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960U-87MG 75% 40–50% SE V4XC-1012 V4XC-1024 V4XC-1032 V4SC-1096 V4SC-1960U-937 36% 85% SG V4XC-3012 V4XC-3024 V4XC-3032 V4SC-3096 V4SC-3960Vero 92% 80–95% SF V4XC-2012 V4XC-2024 V4XC-2032 V4SC-2096 V4SC-2960232North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Line Optimization Kits for 4D Nucleofector,96-well Shuttle and 384-well Nucleofector SystemsThe Cell Line Optimization Nucleofector Kits are the idealtool to conveniently and rapidly determine the optimalNucleofection Condition of virtually any difficult-totransfectcell line <strong>with</strong>in one experiment.With the unique capability of the different NucleofectorPlatforms (4D-Nucleofector System, 96-well ShuttleSystem or 384-well Nucleofector System) to addressindividual wells of a 16-well, 96-well or 384-wellNucleocuvette Plate <strong>with</strong> different programs, cell lineoptimizations are easily performed <strong>with</strong>in one experiment.In each system our three Cell Line Nucleofector SolutionsSE, SF and SG are tested <strong>with</strong> a pre-selected set of programsplus controls.■■Benefits––Optimal Nucleofection Conditions determined on oneplatform are transferable to the others and also to the100 µl single Nucleocuvette on the 4D-NucleofectorX Unit■■Application––Convenient and rapid determination of optimalNucleofection Conditions for virtually any difficult-totransfectcell line <strong>with</strong>in 1 experimentPlatform 4D-Nucleofector System 96-well Shuttle System 384-well Nucleofector System4Kit contents- Four 16-well Nucleocuvette Strips- Specific Nucleofector Solution- Supplement- pmaxGFP Control Vector- One 96-well Nucleocuvette Plate- Specific Nucleofector Solution- Supplement- pmaxGFP Control VectorNumber of optimization reactions 48 rxn (plus 16 rxn for optional fine tuning) 96 rxn 384 rxnOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description Size- One 384-well Nucleocuvette Plate- Specific Nucleofector Solution- Supplement- pmaxGFP Control Vector4D-Nucleofector KitsV4XC-9064 V4XC-9064 Cell Line Optimization 4D-Nucleofector X Kit 20 μl Nucleocuvette 64 reactions (16-well)96-well Shuttle KitsV4SC-9096 V4SC-9096 Cell Line Optimization 96-well Nucleofector Kit 20 μl Nucleocuvette 96 reactions (96-well)384-well Nucleofector KitsV5SC-9001 V5SC-9001 Cell Line Optimization 384-well Nucleofector Kit 20 μl Nucleocuvette 384 reactions (384-well)Transfection / Nucleofector Kits for Cell LinesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com233
Cell Line Kits for Nucleofector II /2b DeviceFor the transfection of cell lines <strong>with</strong> the Nucleofector II/2bDevice, we offer five different Cell Line NucleofectorSolutions: C, L, R, T, and V. Optimized Protocols outlining theoptimal Nucleofector Kit for a large selection of cell linesare available and can be downloaded from our website.■■Benefits––Achieve transfection efficiencies of up to 90% <strong>with</strong> highcell viability4Transfection / Nucleofector Kits for Cell Lines■■Applications––Get up to 99% transfection efficiency <strong>with</strong> siRNAduplexes even in suspension cells––Expression <strong>with</strong>in hours – from transfection to analysisin a daywww.lonza.com/celldatabasewww.lonza.com/protocolsOrdering Information – KitsCat. No. NA Cat. No. EU Product Name SizeVACA-1004 VACA-1004 Cell Line Nucleofector Kit C 10 reactionsVCA-1004 VCA-1004 Cell Line Nucleofector Kit C 25 reactionsVVCA-1004 VVCA-1004 Cell Line Nucleofector Kit C 4 × 25 reactionsVACA-1005 VACA-1005 Cell Line Nucleofector Kit L 10 reactionsVCA-1005 VCA-1005 Cell Line Nucleofector Kit L 25 reactionsVVCA-1005 VVCA-1005 Cell Line Nucleofector Kit L 4 × 25 reactionsVACA-1001 VACA-1001 Cell Line Nucleofector Kit R 10 reactionsVCA-1001 VCA-1001 Cell Line Nucleofector Kit R 25 reactionsVVCA-1001 VVCA-1001 Cell Line Nucleofector Kit R 4 × 25 reactionsVACA-1002 VACA-1002 Cell Line Nucleofector Kit T 10 reactionsVCA-1002 VCA-1002 Cell Line Nucleofector Kit T 25 reactionsVVCA-1002 VVCA-1002 Cell Line Nucleofector Kit T 4 × 25 reactionsVACA-1003 VACA-1003 Cell Line Nucleofector Kit V 10 reactionsVCA-1003 VCA-1003 Cell Line Nucleofector Kit V 25 reactionsVVCA-1003 VVCA-1003 Cell Line Nucleofector Kit V 4 × 25 reactionsQuick Reference Guide – Optimized Protocols for Nucleofector II/2b Device – Cell LinesCell line Efficiency Viable cells Solution 10 rxn 25 rxn 100 rxn293 84% V VACA-1003 VCA-1003 VVCA-100332D 79% 61% V VACA-1003 VCA-1003 VVCA-10033T3-L1 ad 25% 90% L VACA-1005 VCA-1005 VVCA-10053T3-L1 pre-ad 73% 59% V VACA-1003 VCA-1003 VVCA-1003A-10 64% 74% L VACA-1005 VCA-1005 VVCA-1005A-375 72% 97% V VACA-1003 VCA-1003 VVCA-1003A-431 45% 83% T VACA-1002 VCA-1002 VVCA-1002A20 37–74% 81–95% V VACA-1003 VCA-1003 VVCA-1003A2058 81% 94% C VACA-1004 VCA-1004 VVCA-1004A549 72% 81% T VACA-1002 VCA-1002 VVCA-1002A7r5 49% 81% V VACA-1003 VCA-1003 VVCA-1003AGS 73% 62% V VACA-1003 VCA-1003 VVCA-1003ARPE-19 83% 92% V VACA-1003 VCA-1003 VVCA-1003234North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Line Kits for Nucleofector II /2b DeviceContinuedQuick Reference Guide – Optimized Protocols for Nucleofector II/2b Device – Cell LinesCell line Efficiency Viable cells Solution 10 rxn 25 rxn 100 rxnB16-F0 84% 91% R VACA-1001 VCA-1001 VVCA-1001B16-F10 91% 96% V VACA-1003 VCA-1003 VVCA-1003BA/F3 88% 79% V VACA-1003 VCA-1003 VVCA-1003BHK-21 85% 78% L VACA-1005 VCA-1005 VVCA-1005BJ 52% 76% R VACA-1001 VCA-1001 VVCA-1001BxPC-3 28% 62% L VACA-1005 VCA-1005 VVCA-1005C2C12 82% 93% V VACA-1003 VCA-1003 VVCA-1003C6 94% 75–80% V VACA-1003 VCA-1003 VVCA-1003Caco-2 59% 70% T VACA-1002 VCA-1002 VVCA-1002Capan-1 29% 78% V VACA-1003 VCA-1003 VVCA-1003CCRF-CEM 68% 79% C VACA-1004 VCA-1004 VVCA-1004CHO (suspension) 92% 82% V VACA-1003 VCA-1003 VVCA-1003CHO-K1 94% 53–87% T VACA-1002 VCA-1002 VVCA-1002CHO-S (suspension) 90–98% 67–72% V VACA-1003 VCA-1003 VVCA-1003COS-1 49% 64% T VACA-1002 VCA-1002 VVCA-1002COS-7 99% 94% R VACA-1001 VCA-1001 VVCA-1001D1 ORL UVA 61% 97% T VACA-1002 VCA-1002 VVCA-1002DU 145 47% 89% L VACA-1005 VCA-1005 VVCA-1005EL4 65% 76% L VACA-1005 VCA-1005 VVCA-1005FDC-P1 82% 84% L VACA-1005 VCA-1005 VVCA-1005GH3 77% 84% L VACA-1005 VCA-1005 VVCA-1005H9c2(2-1) 86% 90% L VACA-1005 VCA-1005 VVCA-1005HaCaT 43% V VACA-1003 VCA-1003 VVCA-1003HCT 116 78% 76% V VACA-1003 VCA-1003 VVCA-1003HeLa 70% R VACA-1001 VCA-1001 VVCA-1001HeLa S3 67% 95% L VACA-1005 VCA-1005 VVCA-1005Hep G2 41–64% 86–94% V VACA-1003 VCA-1003 VVCA-1003HL-60 90% 50–65% V VACA-1003 VCA-1003 VVCA-1003HT-1080 74% 76% T VACA-1002 VCA-1002 VVCA-1002HT-29 16–51% 57–94% R VACA-1001 VCA-1001 VVCA-1001HuT 78 53% 64% R VACA-1001 VCA-1001 VVCA-1001HUV-EC-C 75% 77% V VACA-1003 VCA-1003 VVCA-1003IMR-32 80% 62% L VACA-1005 VCA-1005 VVCA-1005IMR-90 51% 70% R VACA-1001 VCA-1001 VVCA-1001Jurkat 65–80% 74% V VACA-1003 VCA-1003 VVCA-1003K-562 79% 89% V VACA-1003 VCA-1003 VVCA-1003KG-1 70% 84% R VACA-1001 VCA-1001 VVCA-1001KG-1a 86% 79% L VACA-1005 VCA-1005 VVCA-1005L-428 78% 73% L VACA-1005 VCA-1005 VVCA-1005L6 59% 92% R VACA-1001 VCA-1001 VVCA-1001LNCaP 82% 70–80% R VACA-1001 VCA-1001 VVCA-1001MCF7 77% 60% V VACA-1003 VCA-1003 VVCA-1003MDA-MB-231 79% 77% V VACA-1003 VCA-1003 VVCA-1003MDA-MB-453 54% 90% C VACA-1004 VCA-1004 VVCA-1004MDA-MB-468 60% 81% V VACA-1003 VCA-1003 VVCA-1003MDBK 59% 96% R VACA-1001 VCA-1001 VVCA-1001MDCK 73% 83% L VACA-1005 VCA-1005 VVCA-1005MDCK II 80% 88% L VACA-1005 VCA-1005 VVCA-10054Transfection / Nucleofector Kits for Cell LinesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com235
Cell Line Nucleofector II/2b KitsContinued4Transfection / Nucleofector Kits for Cell LinesQuick Reference Guide – Optimized Protocols for Nucleofector II/2b Device – Cell LinesCell line Efficiency Viable cells Solution 10 rxn 25 rxn 100 rxnMEG-01 80% 66% C VACA-1004 VCA-1004 VVCA-1004MG-63 62% 90% C VACA-1004 VCA-1004 VVCA-1004MOLT-4 55% 61% L VACA-1005 VCA-1005 VVCA-1005MV-4-11 29% 79% L VACA-1005 VCA-1005 VVCA-1005NALM-6 64% 87% T VACA-1002 VCA-1002 VVCA-1002NB-4 71% 66% V VACA-1003 VCA-1003 VVCA-1003NCI-H1299 (H1299) 99% 75% C VACA-1004 VCA-1004 VVCA-1004NCTC clone 929 67% 91% V VACA-1003 VCA-1003 VVCA-1003Neuro-2a (N2a) V VACA-1003 VCA-1003 VVCA-1003NG108-15 64% 82% V VACA-1003 VCA-1003 VVCA-1003NIH/3T3 84% 87–89% R VACA-1001 VCA-1001 VVCA-1001NK-92 26% 40% R VACA-1001 VCA-1001 VVCA-1001NRK 44% 91% T VACA-1002 VCA-1002 VVCA-1002NS0 83% 54% C VACA-1004 VCA-1004 VVCA-1004NTERA-2 cl.D1 90% 94% L VACA-1005 VCA-1005 VVCA-1005P19 85% 80% V VACA-1003 VCA-1003 VVCA-1003P815 62% 92% T VACA-1002 VCA-1002 VVCA-1002PANC-1 68% 75% R VACA-1001 VCA-1001 VVCA-1001PC-12 92% 81% V VACA-1003 VCA-1003 VVCA-1003PC-3 88% 59–66% V VACA-1003 VCA-1003 VVCA-1003Raji 84% 67–81% V VACA-1003 VCA-1003 VVCA-1003Ramos 27% 72% V VACA-1003 VCA-1003 VVCA-1003RAW 264.7 65% 74% V VACA-1003 VCA-1003 VVCA-1003RBL-1 83% 67% V VACA-1003 VCA-1003 VVCA-1003RBL-2H3 42% 93% T VACA-1002 VCA-1002 VVCA-1002S49 81% 68–95% V VACA-1003 VCA-1003 VVCA-1003Saos-2 82% 79% V VACA-1003 VCA-1003 VVCA-1003Schneider’s Drosophila Line 2 77% 64–70% V VACA-1003 VCA-1003 VVCA-1003Sf9 82% 76–79% R VACA-1001 VCA-1001 VVCA-1001SH-SY5Y 63–82% 40% V VACA-1003 VCA-1003 VVCA-1003SK-BR-3 50% 94% C VACA-1004 VCA-1004 VVCA-1004SK-N-SH 85% 73% V VACA-1003 VCA-1003 VVCA-1003SK-OV-3 89% 53% V VACA-1003 VCA-1003 VVCA-1003SW480 60% 86% V VACA-1003 VCA-1003 VVCA-1003T-47D 51% 94% V VACA-1003 VCA-1003 VVCA-1003T/C-28 a2 90% 80% V VACA-1003 VCA-1003 VVCA-1003T/G HA-VSMC 58% 79% V VACA-1003 VCA-1003 VVCA-1003T2 60% 68% C VACA-1004 VCA-1004 VVCA-1004T84 53% 83% T VACA-1002 VCA-1002 VVCA-1002TF-1 38% 82% T VACA-1002 VCA-1002 VVCA-1002THP-1 47–68% 40–58% V VACA-1003 VCA-1003 VVCA-1003U-2 OS 98% 88% V VACA-1003 VCA-1003 VVCA-1003U-87 MG 43% 91% T VACA-1002 VCA-1002 VVCA-1002U-937 20–30% C VACA-1004 VCA-1004 VVCA-1004U266B1 86% 91% V VACA-1003 VCA-1003 VVCA-1003Vero 79% 97% V VACA-1003 VCA-1003 VVCA-1003WEHI-231 77% 62% L VACA-1005 VCA-1005 VVCA-1005WI-38 75% 91% R VACA-1001 VCA-1001 VVCA-1001236North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Line Optimization Kit for Nucleofector II/2b DeviceThe Cell Line Optimization Nucleofector Kit is the ideal toolfor the transfection of virtually any difficult-to-transfect cellline <strong>with</strong> the Nucleofector II/2b Device. It enables you toconveniently determine the optimal NucleofectionCondition of your cell line of interest <strong>with</strong>in one experiment.The kit contains two different Cell Line NucleofectorSolutions, V and L, each of which should be tested incombination <strong>with</strong> seven different Nucleofector Programs.Fine-tuning for optimal results can then be performedtogether <strong>with</strong> our Scientific Support Team.■■Benefits––Efficient transfection of virtually any difficult-totransfectcell line––Simple and rapid optimization completed <strong>with</strong>in justone experiment■■Applications––Transfection of virtually any difficult-to-transfect cellline <strong>with</strong> the Nucleofector II/2b Device1 Solution LV2 Solution VProgram 1 A-020 A -020Program 2 T-020 T-020Program 3 T-030 T -030Program 4 X-001 X -001Program 5 X-005 X -005Program 6 L-029 L-029Program 7 D-023 D -023Step 1The cell line of interest is transfected <strong>with</strong> theNucleofector Solutions L and V in combination <strong>with</strong>seven different Nucleofector Programs.Step 2The Nucleofector Solution and Program which resultin highest transfection efficiencies <strong>with</strong> lowestmortality are selected.> > > +Related ProductsPageClassical Media 100-1113> > >Step 3A further fine tuning of the Nucleofection Conditionscan be performed <strong>with</strong> the help of our ScientificSupport Team.Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description SizeVCO-1001N VCO-1001N Cell Line Optimization Nucleofector Kit 18 reactions4Transfection / Nucleofector Kits for Cell LinesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com237
Basic Parasite Nucleofector Kits4Parasitic protozoa infect vertebrates and invertebrates andsome are even parasitic in plants. In humans, they cancause severe diseases, such as Malaria (Plasmodium),Sleeping Sickness (Trypanosoma) or Leishmaniasis(Leishmania). Nucleofection has proven to provideconsiderably higher transfection efficiencies (e.g., inPlasmodium berghei and Trypanosoma brucei) comparedto standard methods, such as electroporation or particlebombardment. Due to significant genotypic and phenotypicdiversity between species and life cycles, we havedeveloped two Basic Parasite Nucleofector Kits (1 and 2)and an easy-to-use Basic Parasite Nucleofector Starter Kit.■■Benefits––Increased transfection efficiencies compared tostandard methods, such as electroporation or particlebombardmentNucleofection of the rodent malaria parasite Plasmodium berghei.Plasmodium berghei parasites were transfected <strong>with</strong> a reporter vectorcontaining two genes encoding for green fluorescent protein (GFP) andred fluorescent protein (RFP) under control of sex-specific promoters. Afterselection of transgenic parasites, sexual cells (gametocytes) of theseparasites were analyzed by fluorescence microscopy. Male cells showedgreen and female cells a red fluorescence. (Data kindly provided by ChrisJanse, Blandine Franke-Fayard and Andrew Waters, Leiden Malaria<strong>Research</strong> Group, Department of Parasitology, Leiden University MedicalCentre, Netherlands.)Transfection / Nucleofector Kits for Parasites■■Applications––Proven results for Plasmodium berghei andTrypanosoma bruceiOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description SizeVVMI-1011 VVMI-1011 Basic Parasite Nucleofector Kit 1 4 × 25 reactionsVMI-1011 VMI-1011 Basic Parasite Nucleofector Kit 1 25 reactionsVVMI-1021 VVMI-1021 Basic Parasite Nucleofector Kit 2 4 × 25 reactionsVMI-1021 VMI-1021 Basic Parasite Nucleofector Kit 2 25 reactionsVAMI-1011 VAMI-1011 Basic Parasite Nucleofector Kit 1 10 reactionsVAMI-1021 VAMI-1021 Basic Parasite Nucleofector Kit 2 10 reactionsVMI-1001 VMI-1001 Basic Parasite Starter Nucleofector Kit 10 reactions238North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector Kit AccessoriesNucleofector Kit AccessoriesIntroduction240Nucleofector PLUS Supplements 241Mouse T Cell Nucleofector Medium 242pmaxCloning Vector 2434Transfection / Nucleofector Kit AccessoriesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com239
IntroductionWe offer a range of accessory products that can be used incombination <strong>with</strong> our Nucleofector Technology.––Nucleofector PLUS Supplements –For cryopreservationof cells in Nucleofector Solution––Mouse T Cell Nucleofector Medium – For optimalNucleofection Performance <strong>with</strong> mouse T cells––pmaxCloning Vector – For cloning your gene of interestinto a high expression level plasmid4Transfection / Nucleofector Kit Accessories240North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleofector PLUS SupplementsThe new Nucleofector PLUS Supplements bypass the needof postponing a transfection experiment due to problems<strong>with</strong> cell culture or primary cell isolation. Moreover theyallow for a more efficient time-management as freshlyisolated or cultured cells do not have to be transfected atthe day of isolation or harvesting.Nucleofector PLUS Supplements can be used in conjunction<strong>with</strong> almost all existing Nucleofector Kits. Just substitutethe standard supplement (delivered <strong>with</strong> regularNucleofector Kit) for the appropriate Nucleofector PLUSSupplement and generate your own cryopreserved readyto-transfectcompetent cells.■■Benefits––Minimize variations caused by donor variance, isolationprocess or cell culture––More convenient workflow by decoupling of cell isolationand transfection––Cryopreserved transfection competent cells readyto-useany time%1009080706050403020100FreshEfficiencyNHDF-neoFrozenViabilityFreshHUVECFrozen■■Applications––Cryopreservation of primary cells or cell lines inNucleofector Solution––Transfection experiment series <strong>with</strong> the same cell batchComparison of transfection performance for fresh cells and cells that were cryopreserved in Nucleofector PLUS Solution. Data were collected from variousexperiments to account for variances in cell handling.FreshHeLaFrozenFreshNHEK-neoOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description SizeVS1-00150P VS1-00150P Nucleofector PLUS-1 Supplement Suited for: Cell line kits SE, SF, SG, R, T, V, L and Primary cell kits: 150µlP1, P2, P3, P4, and Nucleofector II/2b Kits*VS1-00500P VS1-00500P Nucleofector PLUS-1 Supplement Suited for: Cell line kits SE, SF, SG, R, T, V, L and Primary cell kits: 500µlP1, P2, P3, P4, and Nucleofector II/2b Kits*VS2-00500P VS2-00500P Nucleofector PLUS-2 Supplement Suited for: Cell line kits: C500µlPrimary cell kits: Nucleofector II/2b Kits*VS3-00150P VS3-00150P Nucleofector PLUS-3 Supplement Suited for: Primary cell kits: P5, and Nucleofector II/2b Kits* 150µlVS3-00500P VS3-00500P Nucleofector PLUS-3 Supplement Suited for: Primary cell kits: P5, and Nucleofector II/2b Kits* 500µlVS4-00500P VS4-00500P Nucleofector PLUS-4 Supplement Suited for: Primary cell kits: Nucleofector II/2b Kits* 500µl*To determine the supplement required for your Primary Cell Nucleofector II/2b Kit please refer to the table provided on our website: www.lonza.com/n-plusFrozen4Transfection / Nucleofector Kit AccessoriesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com241
Mouse T Cell Nucleofector MediumFor optimal Nucleofection Performance <strong>with</strong> mouse T cellsit is highly recommended to use Mouse T Cell NucleofectorMedium for cell culture steps post Nucleofection.The medium is included in our Mouse T Cell Nucleofector Kit(for the Nucleofector II/2b Device), and offered as separateproduct when using the P3 Kit <strong>with</strong> the 4D-NucleofectorSystem or the 96-well Shuttle Device.■■Benefits––Provides consistent, high-yield Nucleofection Results––Essential for survival of transfected mouse T cells4■■Applications––For use in combination <strong>with</strong> the P3 Primary Cell4D-Nucleofector or 96-well Nucleofector Kits––For post Nucleofection Culture of mouse T cellsTransfection / Nucleofector Kit AccessoriesOrdering Information – MediumCat. No. NA Cat. No. EU Product Name Size Storage ConditionsVZB-1001 VZB-1001 Mouse T Cell Nucleofector Medium 100 ml 4°C–8°C, do not freezeRelated ProductsMouse B Cell Nucleofector Kits 188Page242North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
pmaxCloning Vector■■Benefits :––High expression rate in mammalian cells––License-free use for research purposes––Multiple cloning site for convenient insertion of thegene-of-interestNOTE: The CMV promoter is covered under U.S. patent 5,385,839 and itsuse is permitted for research purposes only. Any other use of the CMVpromoter requires a license from the University of Iowa <strong>Research</strong>Foundation, 214 Technology Innovation Center, Iowa City, IA.pmaxCloning VectorMCSKpn lPme lHind IIIEcoR IXba lEcoR VPst lBamH lXho lNhe lNot lPme lSac lOrdering Information – KitsCat. No. NA Cat. No. EU Product Name Product Description SizeVDC-1040 VDC-1040 pmaxCloning Vector Concentration: 0.5 µg/µl 20 µg4Transfection / Nucleofector Kit AccessoriesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com243
Notes4Transfection244North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>5 BioAssay Products and Services5Bioluminescent Cell Health 247Cell Function 253
BioAssay Products and ServicesBioluminescent Cell HealthIntroduction248ViaLight Cell Proliferation and Cytotoxicity BioAssay Kit249ToxiLight Non-destructive Cytotoxicity BioAssay Kit 251Cell FunctionIntroduction254PDELight HTS cAMP Phosphodiesterase Assay Kit 255PPiLight Inorganic Pyrophosphate Assay 257AdipoRed Assay Reagent 259AdipoLyze Lipolysis Detection Assay 260OsteoAssay Human Bone Plate 261OsteoLyse Assay Kit 262OsteoImage Mineralization Assay 263BioAssay Accessory Products 2645BioAssays / Cell Health246North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bioluminescent Cell HealthBioluminescent Cell HealthIntroduction248ViaLight Cell Proliferation and Cytotoxicity BioAssay Kit249ToxiLight Non-destructive Cytotoxicity BioAssay Kit 2515BioAssays / Cell HealthEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com247
IntroductionAchieve outstanding sensitivity for cell proliferation and celldeath <strong>with</strong> our easy-to-use bioluminescent cell healthassays. These assays are suitable for use <strong>with</strong> adherent orsuspension cultures of cell lines and primary cells.ViaLight Cell Proliferation and Cytotoxicity BioAssay Kitsare designed to provide unprecedented speed andsensitivity for cytotoxicity and cell proliferation studies, andare safer than traditional radioactive methods.The ToxiLight Non-destructive Cytotoxicity BioAssay Kit isa bioluminescent, non-destructive cytolysis assay kitdesigned to measure the release of the enzyme adenylatekinase (AK) from damaged cells.5BioAssays / Cell Health248North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ViaLight Cell Proliferation and Cytotoxicity BioAssay KitViaLight Plus Cell Proliferation and Cytotoxicity BioAssayKits to provide unprecedented speed and sensitivity forcytotoxicity and cell proliferation studies. Ideal for adherentcells and safer than traditional radioactive methods,ViaLight protocols are as fast and easy other other viabilitykits <strong>with</strong>out any shaking needed. The ViaLight Kitincorporates bioluminescent detection of cellular ATP as ameasure of viability. It delivers high, stable luminescentsignals for an extended period of time, delivering greaterexperimental design flexibility. The easy, two-step,homogeneous assays are scalable for high-throughputapplications in both 96- and 384-well formats, and supporton a variety of luminometers or scintillation counters.The ViaLight MDA Plus Microbial Proliferation andCytotoxicity Kit has been optimized for use <strong>with</strong> bacteria oryeast. The basic reaction remains the same as the ViaLightPlus Kit, however, the lysis reagent has been optimized forbacteria and yeast. Sensitivity is 1,000 bacterial cells or100 yeast cells per well.■■Benefits––Fast – Results from a 96-well plate can be processedand analyzed in
ViaLight Cell Proliferation and Cytotoxicity BioAssay KitsContinuedViaLight Plus BioAssay Kit Sensitivity andExtended LinearityLuminescence (RLU)100,00010,0001,000100EC 50 Data Generated Using ViaLight Plus Showsws nConsistency Over TimeRLUs4,0003,0002,000510Background0.10.1 10 100 1,000 10,000 100,000Cells/WellT= 1 min T= 1 hourT= 2 hoursT= 3 hours T= 4 hoursT= 5 hoursT= 6 hoursLight output at various times after reagent addition and <strong>with</strong> increasingnumbers of K562 cells demonstrate the exceptional sensitivity anddynamic range delivered by the ViaLight Plus BioAssay Kit.1,0000 10 100[Mitomycin C] µg/ml0h EC 506.40 µg/ml3h EC 506.36 µg/ml1h EC 506.46 µg/ml4h EC 506.35 µg/ml2h EC 506.47 µg/ml5h EC 506.36 µg/mlHepG2 cells were incubated <strong>with</strong> the alkylating agent Mitomycin C for 48hours and then assayed using ViaLight Plus. The experiemental valuesare the mean of eight replicate samples read every hour over a 5 hourperiod. The EC values remain consistent over the 5 hour read period.BioAssays / Cell HealthOrdering Information – BioAssay KitCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeLT07-322 LT07-322 ViaLight MDA Plus Microbial Proliferation and Cytotoxicity2°C–8°C, do not freeze 10,000 testsBioAssay KitLT07-122 LT07-122 ViaLight MDA Plus Microbial Proliferation and Cytotoxicity Kit 2°C–8°C, do not freeze 1,000 testsLT17-221 LT17-221 ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit With 5 white TC plates 2°C–8°C, do not freeze 500 testsLT07-321 LT07-321 ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit 2°C–8°C, do not freeze 10,000 testsLT07-221 LT07-221 ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit 2°C–8°C, do not freeze 500 testsLT07-121 LT07-121 ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit 2°C–8°C, do not freeze 1,000 testsRelated ProductsATP Standard 264Clear Bottom, White Walled TC Plates 264ToxiLight Non-destructive Cytotoxicity BioAssay Kit 252Page250North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ToxiLight Non-destructive Cytotoxicity BioAssay KitThe ToxiLight Non-destructive Cytotoxicity BioAssay Kit isa bioluminescent, non-destructive cytolysis assay kitdesigned to measure the release of the enzyme adenylatekinase (AK) from damaged cells. The enzyme activelyphosphorylates ADP to form ATP and the resulting ATP ismeasured using a bioluminescent luciferase reaction. Asthe level of cytolysis increases, the amount of AK in thesupernatant also increases, which results in emission ofhigher light intensity by the ToxiLight Reagent. There is noneed for cell lysis; measurements can be taken directlyfrom the supernatant.■■Benefits––Highly sensitive – Detect as few as ten cells––Non-destructive – Eliminates the need to lyse cells,allowing multiple tests on the same sample––Simple – Addition of a single reagent directly to cells orsupernatant aliquot––Fast – Results from a 96-well plate can be processedand analyzed in
ToxiLight Non-destructive Cytotoxicity BioAssay KitContinuedKinetics of CytotoxicityLuminescence (RLU)60,00050,00040,00030,00020,00010,00006 12 18 24Time (hours)Samples (20 µl) of culture supernatant from cells treated <strong>with</strong>camptothecin were collected at various times and assayed forcytotoxicity.Identify Dose-dependent Activities in CellsViaLight RLUs70,00060,00050,00040,00030,00020,00010,000000 50 100 250 500 1,000 2,000 5,000Camptothecin (nM)ViaLight PlusToxiLight30,00025,00020,00015,00010,0005,000Comparison of ViaLight Plus and ToxiLight Kits using HUVECs dosed <strong>with</strong>camptothecin. The ATP levels indicated by the ViaLight Plus RLUs reducesteadily in a dose-dependent manner. At the lower drug doses, the AKreleased from the cells is relatively low compared <strong>with</strong> that of the control,only increasing dramatically at the highest drug doses.ToxiLight RLUs5BioAssays / Cell HealthToxiLight BioAssay Kit is Sensitive Down to Ten CellsLuminescence (RLU)120,000100,00080,00060,00040,00020,000010,000 20,000 30,000 40,000 50,000R 2 = 0.9979Cells/wellExceptional sensitivity and wide dynamic range results in exceptionalexperimental flexibility.Adenylate Kinase is Stable Over Three DaysRLUs8,0007,0006,0005,0004,0003,0002,0001,0000Day 1 Day 2 Day 3Days after lysatJurkat cells were seeded at 10 5 cells/ml and immediately lysed using theToxiLight 100% Lysis Reagent. The stability of the released AK wasmeasured at 24 hours, 48 hours, and 72 hours after release <strong>with</strong> nosignificant loss in activity.Ordering Information – BioAssay KitCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeLT17-217 LT17-217 ToxiLight Non-Destructive Cytotoxicity BioAssay Kit With 5 white TC plates 2°C–8°C, do not freeze 500 testsLT07-117 LT07-117 ToxiLight Non-Destructive Cytotoxicity BioAssay Kit 2°C–8°C, do not freeze 1,000 testsLT07-217 LT07-217 ToxiLight Non-Destructive Cytotoxicity BioAssay Kit 2°C–8°C, do not freeze 500 testsLT07-517 LT07-517 ToxiLight 100% Lysis Control Set Sold separately 2°C–8°C, do not freeze 200 tests (10 ml)Related ProductsPageViaLight Plus, 500 test kit 250Clear Bottom, White Walled TC Plates 264252North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell FunctionCell FunctionIntroduction254PDELight HTS cAMP Phosphodiesterase Assay Kit 255PPiLight Inorganic Pyrophosphate Assay 257AdipoRed Assay Reagent 259AdipoLyze Lipolysis Detection Assay 260OsteoAssay Human Bone Plate 261OsteoLyse Assay Kit 262OsteoImage Mineralization Assay 263BioAssay Accessory Products 2645BioAssays / Cell FunctionEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com253
IntroductionThe first group of cell function assays is mainly applied inhigh-throughput screening environments. They arehomogeneous, high sensitivity, luminescence-basedassays for enzyme targets including phosphodiesterases(PDELight) and cylases (PPiLight).The second group of cell function assays measure thespecific activities of different cell types including cell linesand primary cells. Adipocytes, MSCs, and ADSCs can havelipid metabolism measured <strong>with</strong> AdipoRed and AdipoLyze.Bone cells like osteoclasts and osteoblasts, and evendifferentiated MSCs and ADSCs can have bone remodelingmeasured <strong>with</strong> OsteoAssay, OsteoLyse and OsteoImage.5BioAssays / Cell Function254North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PDELight HTS cAMP Phosphodiesterase Assay KitThe PDELight HTS cAMP Phosphodiesterase Assay Kit is ageneric, homogeneous assay designed for use in highthroughputscreening to identify inhibitors ofphosphodiesterase activity and IC 50determinations. Theassay utilizes a robust and highly sensitive luciferasebasedbioluminescent system to quantify the AMP producedfrom the hydrolysis of cyclic AMP by phosphodiesterases.AMP is directly converted to ATP and quantitated as light,<strong>with</strong> nearly a photon of light emitted for every molecule ofATP produced. The assay is sensitive, robust andreproducible. Unlike other phosphodiesterase assays, thePDELight Kit does not require costly radioactivity, the useof beads, modified substrates, or antibodies.■■Benefits––Simple – Only one reagent to add––Generic platform – The same assay can be used for allcAMP dependent phosphodiesterases––Rapid assay – Complete a 384-well plate in less than3 minutes––Sensitivity – Allows for the use of enzyme in 96-, 384-,or 1536-well format––Reproducible and robust – Typical Z´ values >0.8, <strong>with</strong>good, clean hits■■Applications––cAMP dependent phosphodiesterase activity screening––IC 50determinations■■Specifications––AMP range:
PDELight HTS cAMP Phosphodiesterase Assay KitContinuedReproducible, High Signal to Background RatiosLinear Detection of AMPLuminescence (RLU)1,600,0001,750,000800,000Z´ = 0.86S/B = 28.3Luminescence [RLU]10 7 0.01 0.1 1 10 10010 610 510 4400,00010 300 10 20 30 40 50Sample No.Signal (S) and background (B) determinations were assessed using asingle phosphodiesterase demonstrating typical high quality data.Z´ results are typically greater than 0.8 <strong>with</strong> excellent signal to backgroundratios.10 2r 2 = 0.9992AMP cAMP [AMP/cAMP] µMThe PDELight Kit measures the AMP produced as a result ofphosphodiesterase activity. The PDELight Detection Reagent measuresAMP up to 20 µM. The PDELight Detection Reagent is specific for AMP andnot cAMP.5BioAssays / Cell FunctionRapid Screening and IC 50Determinations <strong>with</strong> the PDELight Assay Kit% Inhibition100806040200-20-40MMPX3-IBMXCompoundsLeft Image: A library containing 150 pharmacologically active compoundswas screened using the PDELight Kit. The compounds MMPX and 3-IBMXwere identified as inhibiting phosphodiesterase activity greater than 50%at 10 µM.Luminescence (RLU)1,000,000750,000500,000250,00000.01 0.1 1 10 100 1.0IC 50= 6.2 µMIMBX (µM)Right Image: An IC 50of 6.2 µM was determined for 3-IBMX.Ordering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeLT07-600 LT07-600 PDELight HTS cAMP Phosphodiesterase Assay Kit 2°C - 8°C, do not freeze 500 tests256North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PPiLight Inorganic Pyrophosphate AssayInorganic pyrophosphate (also called diphosphate,pyrophosphoric acid or PPi) is a small diphosphate moleculethat is required as a substrate or is the product formed froma number of different enzymatic reactions. Enzymes thatutilize PPi as a substrate may include phosphotransferasesand pyrophosphatases. Enzymes that generate PPi aremore numerous and may include cyclases, hydrolases andligases.The PPiLight Inorganic Pyrophosphate Assay is anon-radioactive bioluminescent assay for the detection ofinorganic pyrophosphate. In the presence of PPi, thedetection reagent catalyses the conversion of AMP to ATP.The assay uses luciferase, which produces light from thenewly formed ATP and luciferin.■■Benefits––Fast – Measure enzyme activity via pyrophosphateconsumption in 1 hour––Simple – Easy two-step luminescent assay <strong>with</strong> noradioactive substrates, beads or antibodies required––Wide detection range – Linear range from 0.02 µM to10 µM––Sensitive – Sensitive to 0.02 µM––Versatile – Scalable to 96-, 384-, and 1586-well formats■■Applications––Measure activity of phosphotransferases andpyrophosphatases––Measure activity of cyclases, ligases, hydrolases andDNA polymerases■■Specifications––Number of tests: 500 tests in 96- or 384-well plates––PPi range: 0.02 µM–10 µM in a 100 µl sample––Assay time: 1 hour––Operating temperature: Ambient––Reproducibility: r 2 value >0.952°C–8°C, do not freezeOrdering information on the next page.5BioAssays / Cell FunctionEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com257
PPiLight Inorganic Pyrophosphate AssayContinuedLight Produced Directly Proportional to PPi PresentRLUs8,000,000Light Increases Proportionally to PPi ConcentrationRLUs1,600,0006,000,0001,200,0004,000,0002,000,0000300,000200,000100,00000 2.5 5 7.5 100 0.1 0.2 0.3 0.412.5PPi µMTypical linear PPi standard curve using the PPiLight InorganicPyrophosphate Assay. Sensitivity is typically 0.02 µM to 10 µM <strong>with</strong> r 2values >0.95.800,000400,000010 20 30 40 50 60 70Minutes0.156 0.313 0.625 1.25PPi signal increases over time at a steady, proportional rate as PPiconcentration increases. Signal linearity is constant throughout a 1 hourincubation.Linear signal5RLUs1,600,0001,200,000BioAssays / Cell Function800,000400,00000.25 0.5 0.75 1 1.25 1.5PPi µM0 0.9994 10 0.999320 0.999730 0.9996 40 0.999850 0.999960 0.9998The linearity of the signal generated <strong>with</strong> varying concentrations of PPiwas assessed over 1 hour. Linearity is not affected by PPi concentrationincrease.Ordering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeLT07-610 LT07-610 PPiLight Inorganic Pyrophosphate Assay 2°C - 8°C, do not freeze 500 tests258North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
AdipoRed Assay ReagentQuantify Intracellular Lipid AccumulationThe AdipoRed Assay Reagent is designed for assessing theeffect of compounds on the differentiation of pre adipocytesor on lipid utilization in mature adipocytes. The lipophilicAdipoRed Assay Reagent specifically partitions into the fatdroplets of differentiated adipocytes and fluoresces at572 nm.This objective, high-throughput, homogeneous, fluorescence-basedassay quantifies the accumulation ofintracellular triglycerides and provides significantadvantages to drug discovery efforts in the field of obesityand diabetes research. It is more sensitive than othermethods, such as the Oil Red O assay, and is much fasterand easier than Northern and Western blots.■■Benefits––Convenient – Simply replace cell culture medium <strong>with</strong>PBS, add AdipoRed Reagent and read in a standardfluorimeter––Fast – Process an entire 96-well plate in as little as20 minutes––Effective – Provides objective, high-throughputmeasurement of the accumulation of intracellulartriglycerides, <strong>with</strong> high signal-to-noise ratiosQuantitation 100,000 of Intracellular Triglyceride AccumulationFluorescenceFluorescence80,00060,00040,00020,0000Poietics Primary Human Preadipocytes were induced to differentiate andassayed using the AdipoRed Assay Reagent.Inhibition of Adipocyte Differentiation Assayed <strong>with</strong>AdipoRed Assay Reagent10,0008,0006,0002 4 6 8 10Days of Incubation5■■Applications––Differentiation of preadipocytes––Lipid utilizationRoom temperature (15°–30°C)4,0002,0000Day 0 Day 4 Day 4TNFαDay 4MifepristonePoietics Primary Human Preadipocytes were induced to differentiate inthe presence of TNFα, Mifepristone or no inhibitor. Lipid accumulation wasassayed after 4 days in culture.Ordering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizePT-7009 PT-7009 AdipoRed Assay Reagent 15°C - 30°C 5 × 4 mlBioAssays / Cell FunctionRelated ProductsPageAdipoLyze Lipolysis Detection Assay 260Adipose Derived Stem Cells 77Human Mesenchymal Stem Cells, Rat Mesenchymal Stem Cells 87, 90Human Subcutaneous and Visceral Preadipocyte Cells 85PGM-2 Preadipocyte Growth Medium-2 BulletKit 86Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com259
AdipoLyze Lipolysis Detection AssayAdipoLyze Lipolysis Detection Assay is a fast, sensitive,fluorescent in vitro assay used for detecting smallquantities of glycerol in cells undergoing lipolysis. TheAdipoLyze Lipolysis Detection Assay can quantitate invitro lipolysis of adipogenic cell lines and primary cells ofboth subcutaneous and visceral origin.A unique advantage of using this assay is the ability toaccurately detect very low levels (down to 0.44 μM or 0.04μg/ml) of glycerol due to the specificity of the enzymes andthe dynamic range of the recommended standard curve.■■Benefits––Quantatively measures glycerol, a product of theprocess of lipolysis––Completed in
OsteoAssay Human Bone PlateMeasure Osteoclastic Bone ResorptionThe OsteoAssay Human Bone Plate provides a thin layer ofadherent human bone for the culture of primary human ornon-human osteoclasts, osteoclast precursors, andimmortalized cell lines. Cells can be stained <strong>with</strong> standardcytochemical (e.g., TRAP) or immunofluorescenttechniques. Assays for measuring bone resorption and/orenzyme activity can be performed easily by sampling thecell culture supernatant.■■Benefits––Convenient – Ready-to-use plates <strong>with</strong> human bonechips attached to wells eliminates the need for dentineor animal bone slices––Simple – Cells can be seeded onto the surface of theOsteoAssay Plate as used in traditional cell cultureprotocols––Flexible – Can be used <strong>with</strong> a variety of cell types andcell-based assays––Novel – Contains real human bone for more biologicallyrelevant results■■Applications––Bone resorption––Osteoclast precursor differentiation––Osteoclast enzymatic activity-20°CTime Course of in vitro Osteoclast Resorptive ActivityAssayed <strong>with</strong> the OsteoAssay Plate and a TelopeptideEIA KitCollagen telopeptide (nM)400300200100024 hr 48 hr 96 hrControlDifferentiatedThe release of collagen peptides from the OsteoAssay Plate bydifferentiating primary human osteoclasts is linear <strong>with</strong> time. PoieticsOsteoclast Precursors were seeded onto an OsteoAssay Plate at 10,000cells/well and cultured in medium containing M-CSF +/- soluble RANKligand. After 5 days of culture, the medium was renewed. Samples ofsupernatant were harvested after an additional 24, 48 and 96 hours and usedin an EIA assay for a telopeptide.OsteoAssay Plate is Superior to Dentine SlicesCollagen helical peptide (ng/ml)160140120100806040200DifferentiatedControlOsteoAssay plateDentine discComparison of primary human osteoclast function (in vitro bonedegradation) when cultured on an OsteoAssay Plate vs. dentine slices.Poietics Human Osteoclast Precursors were seeded at 10,000 cells/wellin the presence of either M-CSF alone (undifferentiated control) or bothM-CSF and soluble RANK ligand (differentiated) for 5 days. Media wererenewed after 5 days and supernatants were harvested after an additional1 day of culture and assayed for collagen peptides.5BioAssays / Cell FunctionOrdering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizePA-1000 PA-1000 OsteoAssay Human Bone Plate Clonetics Primary Biosensors -20°C 96-wellsRelated ProductsPageHuman Osteoclast Precursors 83OCP Osteoclast Precursor BulletKit 83OsteoImage Mineralization Assay 263OsteoLyse Assay Kit (Human Collagen) 262Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com261
OsteoLyse Assay Kit (Human Collagen)Measure Bone Resorption in Minutes5BioAssays / Cell FunctionThe OsteoLyse Assay Kit provides easy-to-use reagents forquantitatively measuring in vitro osteoclast-mediated bonematrix resorption in a high-throughput format. The kitincludes a 96-well cell culture plate coated <strong>with</strong>7europiumlabeled human Type I collagen, and a bottle ofFluorophore Releasing Agent. Osteoclasts can be seededonto the OsteoLyse Plate using traditional cell culture4protocols. The assay directly measures the release ofeuropiumlabeled collagen fragments (resorptive activity)into the osteoclast cell culture supernatant via timeresolved fluorescence.■■Benefits––Convenient – Human collagen is bound to wells in theplate eliminating the need to purchase bone matricesseparately––Easy-to-use – Cells can be seeded onto the surface ofthe OsteoLyse Plate as used in traditional cell cultureprotocols––Homogeneous – Resorptive activity is easily measuredby simply sampling the cell culture supernatant andcounting via time-resolved fluorescence■■Applications––Osteoporosis––Bone resorption––Osteoclast precursor differentiation––Mature osteoclast enzyme activity––Cancer research: metastasis/collagen degradationA comparisonen rona e ed dofOTRAPt aandFu ctOsteoLysenAssay Kits 0in5Alendronate-mediated Osteoclast FunctionMultinucleated TRAP-positive cells500.40.200 0.12 0.23 0.47 0.94 1.88 3.75 8 150Alendronate (mM)TRAP StainOsteoLysePoietics Primary Human Osteoclast Precursors were seeded onto anOsteoLyse Plate at 10,000 cells/well and differentiated <strong>with</strong> M-CSF andsoluble RANK ligand in the presence of interferon γ. At day 10 of culture, 10μl of supernatant was removed and counted. The red line denotes TRAPdata (day 10 multinucleated TRAP-positive cells/well) while the green linerepresents OsteoLyse Assay data.Human Osteoclast Activity Measured byCollagenRelease Using the OsteoLyse Assay KitActivity (RFU)2502001501001,200,0001,000,000800,000600,000400,000200,0000an Oste L s ay n1.61.41.21.0Day 7 Day 8 Day 9 Day 10Time in CultureDifferentiatedUndifferentiated ControlPoietics Primary Human Osteoclast Precursors were seeded onto anOsteoLyse Plate at 10,000 cells/well and differentiated <strong>with</strong> M-CSF andsoluble RANK ligand. At days 7, 8, 9 and 10 of culture, 10 μl of supernatantwas removed and counted. The blue bars represent counts obtained whenthe precursors were cultured <strong>with</strong> M-CSF only.0.80.6OsteoLyse (RFUX 10 6)Ordering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizePA-1500 PA-1500 OsteoLyse Assay Kit Human Collagen 4°C - 8°C 96-wellsRelated ProductsPageHuman Osteoclast Precursors 83OCP Osteoclast Precursor BulletKit 83OsteoImage Mineralization Assay 263262North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
OsteoImage Mineralization AssayRapid, Flourescent Assay for Bone MineralizationThe OsteoImage Mineralization Assay is a rapid,fluorescent, in vitro assay for assessing bone cellmineralization. The assay can quantitate in vitromineralization by osteogenic stem cells, primaryosteoblasts, and osteoblast-like cell lines. It is based onspecific binding of the fluorescent OsteoImage StainingReagent to the hydroxyapatite portion of bone-like nodulesdeposited by cells. The assay is sufficiently sensitive todetect the time-dependent increases in mineralization indifferentiating osteoblast cultures.Unlike typical histochemical methods, such as von Kossaand Alizarin Red, neither of which is hydroxyapatite specific,the OsteoImage Assay eliminates multiple steps or tediousextraction steps. This latest addition to our line of productsfor bone research helps you to increase the speed,sensitivity and ease of measuring mineralization in yourcell cultures.■■Benefits––Delivers qualitative, visual fluorescent microscopy orquantitative plate reader results––Can be used <strong>with</strong> primary osteoblasts, osteoblast stemcells, and osteoblast cell lines––Measures hydroxyapatite, similar to real bone––Completed in less than 90 minutes, <strong>with</strong>out tediousextractions––Sensitive enough to detect time-dependent increasesin mineralization in differentiating cells––Scalable for use in 6-well up to 96-well plates-20°CSimple ProtocolCulture cells▼Remove media and wash <strong>with</strong> PBS▼Fix cells (20 minutes and wash)▼Add OsteoImage Staining Reagent(incubate 30 minutes)▼Wash three times▼View under fluorescence microscope or read on plate readerWorks <strong>with</strong> Stem Cells, Primary Cells, and Cell LinesRFU (492/520)1,0008006004002000UMR-106, Day 7 Saos-2, Day 11 NHOst, Day 21 hMSC, Day 28UndifferentiatedDifferentiatedOsteoblast cell lines, Clonetics NHOst – Normal Human Osteoblasts, andosteoblast-differentiated Poietics hMSC Human Mesenchymal Stem Cellswere evaluated for mineralization <strong>with</strong> the OsteoImage MineralizationAssay on a 96-well plate reader.Detects Mineralization <strong>with</strong> TimeRFU (492/520)6005004003002001000Day 7 Day 14 Day 21Day of assessmentUndifferentiatedDifferentiatedNHOst – Normal Human Osteoblasts were seeded at 3,200 cells/well in a96-well plate. Cells were cultured as undifferentiated control cells or <strong>with</strong>differentiation factors. Mineralization was quantitated on a plate readerafter staining <strong>with</strong> the OsteoImage Assay on days 7, 14 and 21.5BioAssays / Cell FunctionOrdering Information – Assays and ReagentsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizePA-1503 PA-1503 OsteoImage Mineralization Assay -20°C 5 × 96-wellsRelated ProductsPageHuman Mesenchymal Stem Cells 87Human Osteoblast Cells and Growth Medium 83Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com263
BioAssay Accessory ProductsClear Bottom, White-Walled Tissue Culture PlatesClear Bottom White Walled Tissue Culture Plates are whitewalled96-well plastic plates designed specifically for use<strong>with</strong> any bioluminescent bioassay kit.The ATP Standard is a specialized aqueous preparation ofadenosine triphosphate (ATP) and is primarily intended foruse in research to calibrate ATP assays based on theluciferase bioluminescence technique. Each vial contains5 ml of 10 µM ATP.-20°COrdering Information – LabwareCat. No. NA Cat. No. EU Product Name Product Description SizeLT27-102 LT27-102 Tissue Culture Plates Clear bottom, white walled 25 plates5Ordering Information – KitsCat. No. NA Cat. No. EU Product Name Storage Conditions SizeLT27-008 LT27-008 ATP Standard -20°C 5 mlBioAssays / Cell Function264North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>6 Electrophoresis and Analysis6Nucleic Acid Electrophoresis 267Protein Electrophoresis and Analysis 309
Electrophoresis and Analysis6Nucleic Acid ElectrophoresisIntroduction268AgaroseAgarose Selection Guide 269SeaKem® LE Agarose 270MetaPhor Agarose 271The Highest Resolution Agarose Available 271NuSieve 3:1 Agarose 272NuSieve GTG Agarose 273SeaPlaque GTG Agarose 274SeaKem® GTG Agarose 275SeaPlaque Agarose 276ß-Agarase277SeaKem® Gold Agarose 278InCert Agarose and Megabase DNA Standards 279SeaKem® ME Agarose 280SeaPrep Agarose 280I.D.NA Agarose 281Precast Gels for DNA and RNA Selection Guide 282FlashGel System 283FlashGel System for DNA 283FlashGel System for Recovery 284FlashGel System for RNA 286FlashGel Camera 287FlashGel Specifications 287FlashGel SystemPower Supply 288Reliant Minigels 290Latitude HT Gels 292Latitude Midigels 294PAGEr Gold TBE Precast Gels 295Precast Gels and Related Products for RNA Analysis 296Markers, Stains and BuffersDNA Ladders and Markers 300GelStar® Nucleic Acid Gel Stain 302SYBR® Green Nucleic Acid Gel Stains 303AccuGENE Molecular Biology Buffers 304AccuGENE Electrophoresis Buffers 305Gel Support FilmsGelBond® Film 306GelBond® PAG Film 307GelBond® PAG Support Film 308Protein Electrophoresis and AnalysisIntroduction310Precast GelsNew! PAGEr EX Protein Trial Kits 311PAGEr EX Gels 312ProSieveEX Stains 313Prosieve EX Running and Transfer Buffers 314PAGEr Gold Precast Gels 315Selecting the Best PAGEr Gold Precast Gel 316PAGEr Gold Scouting Kit 316PAGEr Minigel Chamber 317ProSieve Color Protein Markers 318ProSieve Protein Markers 319ProSieve ProTrack Dual Color Protein Loading Buffer 319AccuGENE Protein Electrophoresis Buffers 320ProSieve Blue Protein Staining Solution 320SYPRO® Protein Gel Stains 321SYPRO® Ruby Protein Gel Stain 322SYPRO® Red Protein Gel Stain 322SYPRO® Tangerine Protein Gel Stain 323SYPRO® Ruby Protein Blot Stain 323SYPRO® Protein Gel Stain Photographic Filter 324IsoGel Agarose and Precast IsoGel Agarose IEF Plates325IsoGel Agarose 325Precast IsoGel Agarose IEF Plates 326Agarose for Protein Separation 327Agarose for Protein Separation 328ProSieve 50 Acrylamide Gel Solution 329266North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Nucleic Acid ElectrophoresisFrom the Very Beginning to the Next InnovationNucleic Acid ElectrophoresisIntroduction268AgaroseAgarose Selection Guide 269SeaKem® LE Agarose 270MetaPhor Agarose 271The Highest Resolution Agarose Available 271NuSieve 3:1 Agarose 272NuSieve GTG Agarose 273SeaPlaque GTG Agarose 274SeaKem® GTG Agarose 275SeaPlaque Agarose 276ß-Agarase277SeaKem® Gold Agarose 278InCert Agarose and Megabase DNA Standards 279SeaKem® ME Agarose 280SeaPrep Agarose 280I.D.NA Agarose 281Precast Gels for DNA and RNA Selection Guide 282FlashGel System 283FlashGel System for DNA 283FlashGel System for Recovery 284FlashGel System for RNA 286FlashGel Camera 287FlashGel Specifications 287FlashGel SystemPower Supply 288Reliant Minigels 290Latitude HT Gels 292Latitude Midigels 294PAGEr Gold TBE Precast Gels 295Precast Gels and Related Products for RNA Analysis 296Markers, Stains and BuffersDNA Ladders and Markers 300GelStar® Nucleic Acid Gel Stain 302SYBR® Green Nucleic Acid Gel Stains 303AccuGENE Molecular Biology Buffers 304AccuGENE Electrophoresis Buffers 305Gel Support FilmsGelBond® Film 306GelBond® PAG Film 307GelBond® PAG Support Film 308Electrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com267
IntroductionLonza is the leading innovator and world’s most trustedsupplier of agarose and precast gels. We are experts inprotein and nucleic acid electrophoresis, bringing a stronghistory of innovation and reliability to your most importantresearch. Our well known product brands set the standardin quality, purity and performance for electrophoresis.Covering the most extensive range of applications, ourproducts are optimized for the unique requirements of yourmost critical molecular biology techniques. When ourstandard products do not completely fit your needs, inquireabout our custom capabilities to find a product that does.––SeaKem®, NuSieve and MetaPhor Agarose––FlashGel System––Reliant, Latitude and PAGEr Precast Gels––AccuGENE Buffers––GelBond® Gel Support FilmAgarose Selection Guide6Electrophoresis and Analysis / Nucleic AcidSelecting the best agarose for your application can minimizeopportunity for error, optimize results, and even reducecost. We offer a wide range of agarose types that arespecifically engineered to optimize results byfragment size, sample type and application. The selectiontools below will get you started. The following pages willChoose the Agarose that is Right for YouAgarose and applications20 bp - 800 bp50 bp - 1 kb50 bp - 1 kb200 bp - 25 kb200 bp - 25 kb100 bp - 23 kb100 bp - 23 kb1 kb - 10 Mb0 200 bp 400 bp 600 bp 800 bp 1,000 bp 25,000 bp 10,000,000 bpFragment size rangeSeaPlaque GTG AgaroseSeaPlaque AgaroseSeaKem® GTG AgaroseSeaKem® LE AgaroseNuSieve GTG AgaroseNuSieve 3:1 AgaroseMetaPhor AgaroseSeaKem® Gold AgaroseGTGguide you to the right concentration, buffer and marker touse for best performance in your experiment. If you requireadditional support, visit our online Sourcebook forElectrophoresis.www.lonza.com/sourcebookHigh GelStrengthGTGPerformanceCertifiedGTGGTGLow MeltingTemperatureGTG268North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Agarose Selection GuideAgarose and Compatible TechniquesRecovery methodSeaKem®LESeaKem®GTGSeaPlaqueSeaPlaqueGTGNuSieve3:1NuSieveGTGMetaPhorSeaKem®GoldSeaPrep InCert I.D.naIn-gel reactions ■ ■ß-Agarase ■ ■ ■Phenol/chloroform ■ ■ ■Recovery columns ■ ■ ■ ■ ■ ■ ■ ■ ■Electroelution ■ ■ ■ ■ ■ ■ ■ ■ ■Freeze/squeeze ■ ■ ■ ■ ■ ■ ■ ■ ■BlottingSouthern 1 kb ■Northern 1 kb ■Specialty applicationsViral plaque assays ■ ■Preparation ofmegabase samples■PFGE ■ ■Cell culture ■ ■ ■Encapsulation andembedding of cells■DNA identity testing ■Comet assays ■ ■Our agarose is guaranteed DNase/RNase-freeElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com269
SeaKem® LE AgaroseThe Standard for Routine AnalysisHigh GelStrengthSeaKem® LE Agarose is the ideal multipurpose, molecularbiology grade agarose for any DNA or RNA application.■■Benefits––Wide resolution range – 100 bp to 23 kb––High gel strength – ideal for blotting––Consistent lot-to-lot performance■■Applications––Broad range fragment separation––Southern and Northern blotting––PCR greater than 1 kb––Immunoprecipitation techniques––Baculovirus screening and colony lifts18°C–26°CS ake ® LE g eSeaKem® LE AgaroseFinal agaroseconcentration %1.000.80100 bp – 7,000 bp400 bp – 8,000 bp800 bp – 9,000 bp800 bp – 10,000 bp0 1,000 2,000 5,000 10,000 23,000Fragment size bp1X TBE buffer1X TAE buffer1% SeaKem® LE Agarose Gel in TAE BufferPages 461–466www.lonza.com/sourcebook10 kb –5 kb –– 5 kb– 2 kb61 kb –– 1 kb– 500 bpElectrophoresis and Analysis / Nucleic AcidRelated ProductsLane 1: Hind III digest of lambda DNALane 2: DNA marker 1 to 10 kb (Lonza)Lane 3: 500 bp DNA ladder (Lonza)Ordering Information – SeaKem® LE AgaroseCat. No. NA Cat. No. EU Product Name Storage Conditions Size50001 50001 SeaKem® LE Agarose^^ 18°C–26°C 25 g50002 50002 SeaKem® LE Agarose 18°C–26°C 100 g50000 50000 SeaKem® LE Agarose 18°C–26°C 125 g50004 50004 SeaKem® LE Agarose 18°C–26°C 500 g50005 50005 SeaKem® LE Agarose 18°C–26°C 1 kgAccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302Page270North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
MetaPhor AgaroseThe Highest Resolution Agarose AvailableMetaPhor Agarose offers twice the resolution capability ofstandard agarose for PCR, STR and AmpFLP analysis. Thisintermediate melting temperature agarose rivalspolyacrylamide and is capable of resolving DNA fragmentsdiffering in size by 2% between 20 bp and 800 bp.■■Benefits––Fine separation of fragments 20 bp–800 bp––Rivals the resolution capability of polyacrylamide––Eliminates hazards associated <strong>with</strong> polyacrylamide■■Applications––Small PCR analysis––STR analysis––RT-PCRMetaPhor Agarose3.5% MetaPhor Gel horizontal format8 bp Msp I 4 bpladder digest ladder622 bp –242 bp –238 bp –64 bp –56 bp –32 bp –– 67 bp– 34 bp8% Polyacrylamide vertical format8 bp Msp I 4 bpladder digest ladder■■Performance and Quality Tests––DNA resolution: 4 bp resolution of DNA fragments at200 bp and 16 bp resolution at 800 bp in TBE buffer––Gel background: gel exhibits low backgroundfluorescence after ethidium bromide staining––DNA binding: none detectedeta hor A a oseResolution of DNA Ladders in MetaPhor Agarose4.0020 bp – 130 bp50 bp – 250 bp18°C–26°CPages 461–466www.lonza.com/sourcebookRelated Products0 100 200 300 400 500 800Fragment size bpAccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302Final agaroseconcentration %3.002.0050 bp – 250 bp100 bp – 600 bp100 bp – 600 bp150 bp – 800 bp1X TBE buffer1X TAE bufferDNA ladders <strong>with</strong> 4 bp or 8 bp step sizes were prepared by ligation of Bgl IIlinkers. Aliquots of 0.8 µg of the ladders were separated on a 3.5%MetaPhor Agarose gel in a horizontal format and compared to an 8%polyacrylamide gel run in a vertical format in TBE buffer. The horizontal gel(15 cm × 20 cm and 3.0 mm thick) was run at 6.7 V/cm for 4 hours at 15°C.The vertical gel (10 cm × 20 cm and 1.0 mm thick) was run at 8 V/cm for 2hours.Ordering Information – MetaPhor AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50181 50181 MetaPhor Agarose Effective for separating proteins ≥600 kDa 18°C–26°C 25 g50180 50180 MetaPhor Agarose Effective for separating proteins ≥600 kDa 18°C–26°C 125 g50184 50184 MetaPhor Agarose Effective for separating proteins ≥600 kDa 18°C–26°C 500 gLarger package sizes are available upon request. Please inquire for pricing and availability.PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com271
NuSieve 3:1 AgaroseThe Reliable Choice for PCR AnalysisHigh GelStrengthNuSieve 3:1 Agarose was the first and still is the mostreliable choice for separating and resolving PCR and RT-PCRfragments. This molecular biology grade agarose producesstrong, easy-to-handle gels, making it ideal for blotting ofsmall fragments.■■Benefits––Exceptional resolution of small fragments between50 bp and 1 kb––Superior gel strength for blotting––Widely cited as the choice for PCR analysis■■Applications––Small DNA and RNA fragment analysis––Blotting of small fragments––RT-PCR and Genotyping■■Performance and Quality Tests––Resolution: DNA fragments ≤1,000 bp are finely resolvedafter electrophoresis––Gel background: gel exhibits low backgroundfluorescence after ethidium bromide staining––DNA binding: none detectedPCR Products on a NuSieve 3:1 Agarose Gel1,353 bp –622 bp –404 bp –242 bp238 bp67 bp –34 bp –1 2 3 4 5 6 7 8 9 10PCR ProductPrimer-DimerPrimerA 550 bp sequence from lambda DNA was amplified (25 cycles) usingprimers and Taq DNA polymerase supplied in the GeneAmp® Kit (RocheMolecular Systems). PCR products and controls were electro phoresed on a4% NuSieve 3:1 Agarose gel in TAE buffer at 5 V/cm for 3 hours. Lane 1,Msp I digest of pBR322 DNA (1.5 µg); lane 2, Hae III digest of øX174 DNA(1.5 µg); lane 3, no DNA control; lanes 4–9, PCR products resulting fromdifferent reaction conditions (7 µl of 100 µl reaction mixture); and lane 10,a positive control where kit template was added.618°C–26°CNuSieve 3:1 AgaroseElectrophoresis and Analysis / Nucleic AcidFinal agaroseconcentration %4.003.0050 bp – 400 bp100 bp – 500 bp100 bp – 500 bp0 100 200 300 400 500 1,000Fragment size bp1X TBE buffer1X TAE buffer500 bp – 1,000 bpPages 461–466www.lonza.com/sourcebookOrdering Information – NuSieve 3:1 AgaroseCat. No. NA Cat. No. EU Product Name Storage Conditions Size50091 50091 NuSieve 3:1 Agarose 18°C–26°C 25 g50090 50090 NuSieve 3:1 Agarose 18°C–26°C 125 g50094 50094 NuSieve 3:1 Agarose 18°C–26°C 500 gLarger package sizes are available upon request. Please inquire for pricing and availability.Related ProductsPageAccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302272North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
NuSieve GTG AgarosePerformance Certified for Small Fragment Recovery and In-gel ReactionsLow MeltingTemperatureGTGPerformanceCertifiedNuSieve GTG Agarose provides optimal separation andresolution of PCR and RT-PCR fragments. This low melting(≤65°C) temperature agarose is easy-to-handle and can beused for cloning procedures directly from remelted agarose.Genetic Technology Grade Agarose is quality tested tocertify performance.Fine Resolution of Low Molecular Weight DNAFragments in NuSieve GTG Agarose2%AB3%AB4%AB– 622 bp■■Benefits––Fine resolution of small fragments between 50 bpand 1 kb––Performance certified for digestion and ligation■■Applications––Analysis and recovery of small DNA fragments––In-gel PCR and In-gel ligations/transformations1,353 bp –603 bp –281 + 271 bp –– 242 + 238 bp– 160 bp– 147 bp– 90 bp– 67 bp■■Performance and Quality Tests––Enzymatic activity in the presence of remelted gel:T4 DNA ligase and transformation test––Resolution: DNA fragments ≤1,000 bp are finelyresolved after electrophoresis––Gel background: gel exhibits low backgroundfluorescence after ethidium bromide staining––DNase and RNase activity: none detected––DNA binding: none detected194 bp –118 bp –72 bp –– 34 bpDNA fragments were separated in 2%, 3%, and 4% NuSieve GTG Agarosegels in 1X TBE buffer. Lane A: Hae III digest of øX174 DNA, 0.5 µg/lane. LaneB: Msp I digest of pBR322 DNA, 0.5 µg/lane. Running conditions: 1X TBE at5 V/cm.NuSieve GTG GTG A aro Agarose618°C–26°CRelated ProductsFinal agaroseconcentration %4.003.0050 bp – 400 bp100 bp – 500 bp100 bp – 500 bp0 100 200 300 400 500 1,000Fragment size bp1X TBE buffer1X TAE buffer500 bp – 1,000 bpOrdering Information – NuSieve GTG AgaroseCat. No. NA Cat. No. EU Product Name Storage Conditions Size50081 50081 NuSieve GTG Agarose 18°C–26°C 25 g50080 50080 NuSieve GTG Agarose 18°C–26°C 125 g50084 50084 NuSieve GTG Agarose 18°C–26°C 500 gLarger package sizes are available upon request. Please inquire for pricing and availability.AccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com273
SeaPlaque GTG AgarosePerformance Certified for Large Fragment Recovery and In-gel ReactionsLow MeltingTemperatureGTGPerformanceCertified6Confidently resolve fragments from 200 bp to 25 kb prior toPCR, cloning, digesting, or sequencing in the presence ofre-melted SeaPlaque GTG Agarose, <strong>with</strong>out additionalpurification steps. This low-melting temperature (≤65°C)Genetic Technology Grade Agarose is quality tested tocertify performance.■■Benefits––Optimal separation range for DNA and RNA recovery offragments: 200 bp to 25 kb––Performance certified■■Applications––Analysis and recovery of large DNA fragments––In-gel PCR and In-gel ligations and transformations––DNA and RNA digestion■■Performance and Quality Tests––Enzymatic activity in the presence of remelted gel:––T4 DNA ligase and transformation test––Hind III and EcoR I restriction digestion test––Fine resolution of DNA fragments ≥1,000 bp <strong>with</strong> lowbackground after ethidium bromide staining––DNase and RNase activity: none detected––DNA binding: none detectedResolution Performance of SeaPlaque GTG Agarose12 kb –6 kb –4 kb –2 kb –1 kb –0.5 kb –1X TAE buffer0.75% 1.00% 1.25%1X TBE buffer0.75% 1.00% 1.25%12 kb– 6 kb4 kb– 2 kb– 1 kb– 0.5 kbSeparation of DNA markers in 0.75% to 1.25% SeaPlaque GTG Agarosegels in 1X TAE and TBE buffers. 1 kb DNA ladder, 1 µg/lane, DNA unheatedprior to loading. The gels were cast in a 25.5 cm framing gel of 1% SeaKem®GTG Agarose in a submarine chamber and run under 5 mm of bufferoverlay at 5 V/cm for 3 hours, 40 minutes (TBE buffer) and 4 hours, 30minutes (TAE buffer).SeaPlaque GTG AgaroseElectrophoresis and Analysis / Nucleic Acid18°C–26°CPages 461–466www.lonza.com/sourcebookRelated ProductsFinal agaroseconcentration %1.501.00100 bp – 3,000 bp150 bp – 6,000 bp200 bp – 12,000 bp300 bp – 20,000 bp0 1,000 2,000 5,000 10,000 20,000Fragment size bp1X TBE buffer1X TAE bufferOrdering Information – SeaPlaque AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50111 50111 SeaPlaque GTG Agarose 18°C – 26°C 25 g50110 50110 SeaPlaque GTG Agarose 18°C – 26°C 125 g58001 58001 ß-Agarase 18°C–26°C 100 units58005 58005 ß-Agarase 18°C–26°C 500 unitsLarger package sizes are available upon request. Please inquire for pricing and availability.AccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302Page274North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
SeaKem® GTG AgarosePerformance Certified for Large Fragment RecoveryHigh GelStrengthGTGPerformanceCertifiedSeaKem® GTG Agarose ensures reliable digestion andligation from recovered DNA or RNA fragments from 100 bpto 23 kb. Our Genetic Technology Grade Agarose is qualitytested to certify performance.■■Applications––Best choice for DNA and RNA recovery and cloning offragments 100 bp to 23 kb■■Performance and Quality Tests––Restriction endonuclease digestion test: EcoR I andHind III are tested for complete digestion ofelectroeluted, linearized pBR322 DNA––Ligation of recovered DNA––Fine resolution of DNA fragments ≥1,000 bp <strong>with</strong> lowbackground after ethidium bromide staining––DNase and RNase activity: none detected––DNA binding: none detected18°C–26°CPages 441–443www.lonza.com/sourcebookResolution Performance of SeaKem® GTG AgaroseEfficient Digestions after Recovery% Digest using Hind III100806012 kb –6 kb –4 kb –2 kb –1 kb –0.5 kb –1X TAE buffer0.75% 1.00% 1.25%1 kb DNA ladder (Lonza) 1 µg/lane, unheatedSeaKem® GTG AgaroseGTGFinal agaroseconcentration %1.000.80Related Products100 bp – 7,000 bp400 bp – 8,000 bp800 bp – 9,000 bp800 bp – 10,000 bp0 1,000 2,000 5,000 10,000 23,000Fragment size bp1X TBE buffer1X TAE buffer40200A B C D(A) SeaKem® GTG Agarose, (B) Competitor’s agarose,(C) Competitor’s agarose, (D) Competitor’s agaroseOrdering Information – SeaKem® GTG AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50071 50071 SeaKem® GTG Agarose 18°C–26°C 25 g50070 50070 SeaKem® GTG Agarose 18°C–26°C 125 g50074 50074 SeaKem® GTG Agarose 18°C–26°C 500 gLarger package sizes are available upon request. Please inquire for pricing and availability.AccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com275
SeaPlaque AgaroseThe Original Low-melting Temperature AgaroseLow MeltingTemperatureSeaPlaque Agarose is the original low-melting temperatureagarose and has been a staple in molecular biology labs forover 40 years. This molecular biology grade agaroseproduces gels <strong>with</strong> greater sieving capabilities from 200 bpto 25 kb, and <strong>with</strong> higher clarity than standard meltingtemperature agarose. Ideal for preparative DNA and RNAelectrophoresis.■■Benefits––Ideally suited for DNA and RNA recovery––Also ideal for cloning of tissue culture cells and viralplaque assays■■Applications––Preparative DNA and RNA electrophoresis––Viral plaque assays––Cell culture18°C–26°C1% SeaPlaque Agarose Gel10 kb –2 kb –1 kb –1 2 3 4– 3 kbSeaPlaque Agarose– 1.3 kb– 700 bp– 400 bp– 100 bpLane 1:DNA marker 1 to 10 kb (Lonza)Lane 2:DNA marker 50 to 2,500 bp (Lonza)Lane 3:500 bp DNA ladder (Lonza)Lane 4:100 bp Extended Range DNA ladder (Lonza)6Pages 441–443www.lonza.com/sourcebookFinal agaroseconcentration %1.501.00100 bp – 3,000 bp150 bp – 6,000 bp200 bp – 12,000 bp300 bp – 20,000 bp0 1,000 2,000 5,000 10,000 20,000Fragment size bp1X TBE buffer1X TAE bufferElectrophoresis and Analysis / Nucleic AcidOrdering Information – SeaPlaque AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50101 50101 SeaPlaque Agarose A low melting alternative for separating proteins ≥600 kDa. 18°C–26°C 25 g50100 50100 SeaPlaque Agarose A low melting alternative for separating proteins ≥600 kDa. 18°C–26°C 125 g58001 58001 ß-Agarase 18°C–26°C 100 units58005 58005 ß-Agarase 18°C–26°C 500 unitsLarger package sizes are available upon request. Please inquire for pricing and availability.Related ProductsAccuGENE 1 M Tris HCl Buffer 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302Page276North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ß-AgaraseRecovers DNA and RNA from Low-melting Temperature Agaroseß-Agarase is an enzyme that will completely digest thepoly saccharide backbone of molten agarose into alcoholsoluble oligosaccharides. DNA electrophoresed in lowmelting temperature agarose gels can be recovered quantitativelyafter the gel is melted and then digested <strong>with</strong> thisenzyme. Any remaining agarose oligosaccharides will notgel or interfere <strong>with</strong> subsequent DNA manipulations such ascloning, labeling, restriction digestion, or sequencing.Recovery of DNA from 1% SeaPlaque GTG Agarose<strong>with</strong> ß-AgaraseConcentrationß-Agarase is supplied at 1,000 units/ml in a bufferconsisting of 50% glycerol, 50 mM Tris/HCl, 100 mM NaCl,and 0.1% Triton® X-100 at pH 7.5.Unit DefinitionOne unit contains the amount of enzyme necessary tocompletely digest 200 mg of molten 1% SeaPlaque GTGAgarose gel prepared in 40 mM Bis Tris/HCl, 40 mM NaCl,1 mM EDTA (pH 6.0) at 40°C in 1 hour.Hind III-digested lambda DNA gel stained <strong>with</strong> ethidium bromide and theDNA bands excised. Gel digested <strong>with</strong> ß-Agarase, and DNA recovered byethanol precipitation. The recovered DNA was applied to a 1% SeaKem®GTG Agarose gel in 1X TBE buffer and separated by electro phoresis. Aphotograph of the second gel stained <strong>with</strong> ethidium bromide is shown.■■Performance and Quality Tests––No detectable DNase or RNase18°C–26°Cwww.lonza.com/sourcebookOrdering Information – ß-AgaraseCat. No. NA Cat. No. EU Product Name Storage Conditions Size58001 58001 ß-Agarase 18°C–26°C 100 units58005 58005 ß-Agarase 18°C–26°C 500 unitsLarger package sizes are available upon request. Please inquire for pricing and availability.Related ProductsNuSieve GTG Agarose 273SeaPlaque Agarose 274,276,328SeaPlaque GTG Agarose 274PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com277
SeaKem® Gold AgarosePerformance Certified for Rapid Resolution of Megabase DNA by PFGEHigh GelStrengthGTGPerformanceCertifiedSeaKem® Gold Agarose is ideal for separating very large DNAfragments or doing pulsed field gel electrophoresis (PFGE).This Genetic Technology Grade Agarose is ideal for rapidresolution of megabase DNA, decreasing run times by up to50% for PFGE.■■Benefits––Capable of rapid separation of large DNA from 30 kb to50 kb by horizontal electrophoresis or 50 kb to 10 Mbby PFGE––Good multipurpose, high gel strength agarose forseparations ≥1,000 bp––Specially manufactured to create a strong gel that iseasy-to-handle––Guaranteed DNase and RNase-freePerformance of SeaKem® Gold Agarose for DNA ≤ 50 kb48 kb –19 kb –12 kb –6 kb –4 kb –2 kb –1 kb –0.5 kb –1X TAE buffer0.3% 0.3% 0.5% 0.5%1X TBE buffer0.3% 0.3% 0.5% 0.5%– 48 kb– 19 kb– 12 kb– 8.6 kb– 8.3 kb1 2 3 4 1 2 3 46Electrophoresis and Analysis / Nucleic Acid■■Applications––Large fragment separation––Pulsed field gel electrophoresis––Blotting of megabase DNA■■Performance and Quality Tests––Relative DNA mobility: 1.3 under PFGE conditions(SeaKem® LE Agarose = 1.0)––Restriction endonuclease digestion test: EcoR I andHind III tested for complete digestion of recovered DNA––Ligation of recovered DNA––Resolution: DNA fragments ≥1,000 bp are finely resolvedafter electrophoresis––Gel background: gel exhibits low backgroundfluorescence after ethidium bromide staining––DNase and RNase activity: none detected––DNA binding: none detectedRelated ProductsDNA markers separated in 0.3% and 0.5% SeaKem® Gold Agarose gels in1X TAE and TBE buffers. Lanes 1 and 3 are 1 kb ladders, 1 µg/lane, DNAunheated prior to loading. Lanes 2 and 4 are high molecular weightmarkers (8.3, 8.6, 10.1, 12.2, 15.0, 17.0, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4,48.5 kb), 0.3 µg/lane, DNA heated 10 minutes at 65°C prior to loading. Gelswere cast in a 25.5 cm framing gel of 1% SeaKem® GTG Agarose in asubmarine chamber and run under 5 mm of buffer overlay at 1 V/cm for 16hours (TAE buffer), and 20 hours (TBE buffer).18–26°CPages 461–466www.lonza.com/sourcebookOrdering Information – SeaKem® Gold AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50152 50152 SeaKem® Gold Agarose Effective for separating proteins ≥600 kDa 18°C–26°C 25 g50150 50150 SeaKem® Gold Agarose Effective for separating proteins ≥600 kDa 18°C–26°C 125 gLarger package sizes are available upon request. Please inquire for pricing and availability.InCert Agarose and Megabase DNA Standards 279AccuGENE Buffers 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302Page278North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
InCert Agarose and Megabase DNA StandardsUsed to Prepare Chromosomal DNA for PFGEInCert Agarose is a low-gelling temperature agarose,certified for use in the preparation and digestion ofchromosomal DNA prior to pulsed field gel electrophoresis(PFGE).Performance of Megabase DNA Standards in PFGELambda laddersS. cerevisiaeOur Megabase DNA Standards are specially prepared andtested chromosomal DNA standards offered in InCertAgarose gel plug format for easy handling during PFGE.■■Benefits––Certified performance for chromosomal DNA preparationand restriction endonuclease digestion––Performance tested for reliable PFGE––Saves time – standards are ready-to-use■■Applications––Pulsed field gel electrophoresis■■Performance and Quality Tests for InCert Agarose––Restriction endonuclease digestion in agarose gel plugstest: enzymes tested on E. coli DNA: EcoR I, and Hind III––DNase activity: none detectedLambda DNA ladders and S. cerevisiae and DNA Standards were run on theBio-Rad® CHEF-DR® III System.Running conditions:Lambda ladders: 1% SeaKem® GTG Agarose, 0.5X TBE, switch angle 120°,6 V/cm, ramped switch time from 50–90 seconds over 22 hours.S. cerevisiae: 1% SeaKem® GTG Agarose, 0.5X TBE, switch angle 120°,6 V/cm, ramped switch time from 40–100 seconds over 24 hours.InCert Agarose: 18°C–26°CMegabase DNA Standards: 2°C–8°CPages 461–466www.lonza.com/sourcebookOrdering Information – InCert Agarose and Megabase StandardsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50121 50121 InCert Agarose 18°C–26°C 1 g50123 50123 InCert Agarose 18°C–26°C 5 g50401 50401 Lambda DNA Ladder 50 Kb to 873 Kb 5 plugs(10 ± 2 μg DNA/plug)50411 50411 Saccharomyces cerevisiae DNA Standard 220 kb to approximately 1 Mbchromosomal DNA2°C–8°C5 plugs(10 ± 2 μg DNA/plug)Larger package sizes are available upon request. Please inquire for pricing and availability.Related ProductsAccuGENE Buffers 304PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com279
SeaKem® ME AgaroseIdeal for Serum Protein and IEP AnalysisSeaKem® ME Agarose is the ideal choice for serum proteinelectrophoresis and immunoelectrophoresis, and may beused for DNA electrophoresis.■■Benefits––Enhanced resolution in serum protein electrophoresis––High gel clarity and minimal non-specific binding18°C–26°CPages 461–466www.lonza.com/sourcebook■■Applications––Serum protein electrophoresis––Immunoelectrophoresis––Nucleic acid electrophoresisOrdering Information – SeaKem® ME AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50011 50011 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 18°C–26°C 25 g50010 50010 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 18°C–26°C 125 g50014 50014 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 18°C–26°C 500 gLarger package sizes are available upon request. Please inquire for pricing and availability.6Electrophoresis and Analysis / Nucleic AcidSeaPrep AgaroseIdeal for Cell Culture ApplicationsSeaPrep Agarose is a unique ultra-soft agarose, ideal forhigh efficiency hybridoma cloning. It is also used forexpanding cDNA libraries in a strictly representativefashion, decreasing the possibility that less abundantclones vanish during amplification due to differential ratesof replication.■■Specifications––Melting temp: ≤50°C at 1%––Gelling temp: 8°C–17°C at 0.8%––Gel Strength: >75 g/cm 2 at 2%Related Products■■Applications––Cell culture––Hybridoma cloning––Encapsulation/embedding of cells18°C–26°CPages 461–466www.lonza.com/sourcebookOrdering Information – SeaPrep AgaroseCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50302 50302 SeaPrep Agarose 18°C–26°C 25 gLarger package sizes are available upon request. Please inquire for pricing and availability.AccuGENE Buffers 304Page280North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
I.D.NA AgaroseDesigned for Identity TestingI.D.NA Agarose is specially manufactured for DNA identitytesting. For reliable separation of VNTRs, HVRs, RFLPs, andDNA size standards, it is a perfect match for your DNA typingtests.■■Benefits––Performance certified to assure lot-to-lot reliability forDNA identity testing––Crisp DNA separation to accurately discriminate DNAfragments––Strong, easy-to-handle gels allow for trouble-free highefficiency blotting■■Applications––DNA identity testing■■Performance and Quality Tests––DNase and RNase activity: none detected––DNA binding: none detected18°C–26°CPage 462 (Analytical specifications)Resolution and Transfer Performance of I.D.na Agarose1 2– 23 kb– 10 kb– 5.2 kb– 2.3 kb– 1.3 kbAn autoradiogram of DNA size standards (LIFECODES Corp.) and Hae IIIdigestedK562 DNA probed <strong>with</strong> D4S139 (Invitrogen). DNA waselectrophoresed at 1 V/cm for 16 hours in a 1% I.D.na Agarose gel,transferred, and probed. Lane 1: DNA size standards; Lane 2: alleles detected<strong>with</strong> D4S139.Ordering Information – I.D.NA AgaroseCat. No. NA Cat. No. EU Product Name Storage Conditions Size50170 50170 I.D.NA Agarose 18°C–26°C 125 gRelated ProductsLarger package sizes are available upon request. Please inquire for pricing and availability.AccuGENE Buffers 304DNA Ladders and Markers 301GelStar® and SYBR® Green Nucleic Acid Gel Stains 298,302MetaPhor Agarose 271,328PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com281
Precast Gels for DNA and RNA Selection GuideWe offer a complete family of precast agarose gels for DNAand RNA electrophoresis. Our unique gel options cover thefull range of separations needs, from ultra-fast PCR analysisand recovery, to fine resolution and high-throughputseparations. Our custom manufacturing capabilities canFlashGel Systemsupport the requirements of nearly any application. AllLonza Gels are precision manufactured <strong>with</strong> our high qualitySeaKem® and NuSieve Agarose and functionally tested forconsistent performance.Reliant MinigelsNo. of Wells12 + 1, 2 × 16 + 1and 2 × 8 + 1loading Volume5 µl, 12 µl8.4 cm7.0 cmNo. of Wells20loading Volume
FlashGel SystemFast, Sensitive, Simple Analysis, Recovery, and Documentation of DNA and RNAThe FlashGel System gets straight to your results. Simplyload samples, watch bands migrate and get data in as little as2 minutes. Say goodbye to gel preparation, band excision,purification, and UV light. Complete separation, recovery anddocumentation safely, at the bench, in minutes.■■5 Minute Separation and Recovery––See bands in as little as 2 minutes––Recover samples directly, <strong>with</strong>out UV light, bandexcision or purification■■Real-time Separation and Documentation––Watch band migration as it happens––Photograph gels at the bench, <strong>with</strong>out DNA damagingUV light■■Outstanding Sensitivity and Resolution––5–20 times more sensitive than ethidium bromide;detect
FlashGel System for DNAContinued■■Real-time Visualization––Built-in illumination, allows you to view DNA underambient light as it migrates through the gel; stop the runwhen desired separation is reached; safely view thecassette on the lighted dock <strong>with</strong>out eye protection.––DNA bands separated on FlashGel Cassettes are alsodetectable by UV light and may be photographed usingstandard gel documentation systems. Use the FlashGelCamera for best performance.■■Superior Resolution––Resolve fragments in 2–7 minutes, and see clean, sharpband separation, and straight, uniform sample lanesComparison of FlashGel System <strong>with</strong> Company I1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 121.2% FlashGel Cassette, 12+1 well,single-tier format. 275 V, 7 minuterun on The FlashGel Dock.Company I 1.2% gel, 12 well,single-tier format. 30 minute run.DNA bands as viewed during a run on the FlashGel Dock.Lanes 1 and 7: FlashGel DNA Marker (100 bp–4 Kb);Lanes 2 and 8: FlashGel QuantLadder;Lanes 3 and 9: Lonza 100 bp Ladder;Lanes 4 and 10: Lonza 50–2500 bp Marker;Lanes 5 and 11: 285 bp ß-actin PCR*;Lanes 6 and 12: 294 bp control PCR* (Company A)*Samples diluted <strong>with</strong> 1X FlashGel Loading Dye prior to loading.Page 289www.flashgel.com6Electrophoresis and Analysis / Nucleic AcidPage 287 (specifications)Related ProductsFlashGel System for Recovery 289FlashGel System for RNA 289FlashGel Camera 289FlashGel Dock 289FlashGel Power Supply 288FlashGel System for Recovery5 Minute DNA RecoveryDirect DNA recovery using the FlashGel System forRecovery eliminates agarose gel preparation, band excision,and purification. The system delivers highly efficientrecovery, free from inhibitors and UV-induced damage, in asimple 5–10 minute protocol.––Go from sample loading to recovery in just 5 minutes––Recover samples directly from the cassette, <strong>with</strong>outband excision or purification––Visualize sample recovery <strong>with</strong>out UV––Recover at 80%–100% efficiencywww.flashgel.com■■Fast, Simple ProcedurePage1. Load samples in top tier of wells.2. Run until band of interest almost reaches thesecond tier of wells.3. Stop the run and add FlashGel Recovery Buffer.4. Start and run band of interest into the well.5. Stop the run and remove DNA from well via pipette.284North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
FlashGel System for RecoveryContinued■■No DNA Damaging UV or Mutagenic Stain Exposure––Visible light from the compact FlashGel Dockilluminates the recovery wells <strong>with</strong>out damage to theDNA or hazard to the user––The proprietary stain in the FlashGel Cassettes enablesseparation and recovery of very small quantities ofDNA, and minimizes user exposure to potentialmutagens■■Efficient Recovery, Free from Inhibitors––Samples are recovered at 80%–100% efficiency, arefree of inhibitors, and ready for subsequentre-amplification, cloning, or other techniques, <strong>with</strong>outadditional clean-up stepsDNA Size Range on the FlashGel System for RecoveryRecovery Efficiency on the FlashGel System forRecovery1. Recovered Samples 2. Plasmid Restriction DigestsFG C FG1 FG2 C1 C2 VPlasmid DNA (pBr322) was subjected to restriction enzyme doubledigestion using PstI and BamHI. Samples of the restricted DNA wereseparated and 3.2 kb fragments were recovered using the FlashGelRecovery System (FG) or spin column kits (C1 and C2). Image 1 compares5% of each recovered sample. Aliquots of the recovered samples wereligated into PstI/BamHI double digested pUC19 vector (V). Samples of theligation reactions were transformed into E.coli competent cells. Thenumber of colonies obtained <strong>with</strong> both samples were very similar. Image 2shows examples of PstI/BamHI cut plasmid samples from two coloniesfrom each sample. V shows a restricted sample of vector <strong>with</strong> no insert.Samples were separated and recovered on a FlashGel Recovery Cassette.3 µl aliquots of recovered samples consisting of 100 ng of fragmentsranging from 50 bp to 4000 bp separated on a 1.2% FlashGel DNA Cassetteand compared to the FlashGel DNA Marker 100 bp–4 kb and the FlashGelQuantLadder.Page 289Page 287 (specifications)www.flashgel.comRelated ProductsFlashGelRecovery SystemSeparate fragments onFlashGel System3–5 minutesRecover DNAdirectly from wells1–5 minutesColumn RecoveryMethodSeparate fragments onagarose gel30–60 minutesExcise bands fromagarose gel5–10 minutesRecover DNA viaspin column25–30 minutesFlashGel System for DNA 289FlashGel System for RNA 289FlashGel Camera 289PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com285
FlashGel System for RNARapid, Sensitive, Convenient RNA AnalysisThe FlashGel System for RNA is the ideal tool for rapidverification of sample integrity prior to downstreamanalysis. High quality, intact RNA is essential for consistentresults in gene expression, Northern analysis, cDNA libraryconstruction and cDNA labeling for microarrays.Separation of Total RNA on theFlashGel System for RNA1 2 3 4––Get results in 30 minutes or less––Detect
FlashGel CameraFrom Benchtop to Desktop in 5 MinutesCapture data from The FlashGel System and say goodbye todarkrooms and UV light. Complete separation anddocumentation safely, at your bench in minutes. This simpledigital camera in an enclosed hood connects directly to yourlaptop or PC via USB. Simply click a button to capture thedesired image to a file.■■Real time Separation and Documentation––Complete gel run and image capture in just 5 minutes––Photograph gels at the bench <strong>with</strong>out UV light■■The FlashGel Camera Offers––Sharp, clear high-resolution images––Simple user interface––Small, compact design––Optimized exposure for FlashGel CassettesPage 289www.flashgel.comCamera SpecificationsHood dimensions: 10 cm (W) × 11 cm (L) × 16 cm (H)Camera type:DigitalImage file type:.jpg, .tif, .bmpRelated ProductsPageFlashGel System for DNA 289FlashGel System for RNA 289FlashGel System for Recovery 289FlashGel SpecificationsSimple User Interface Right from Your Laptop or PCCassette and Dock SpecificationsOptimal separationand recoveryrange:DNA: 1.2% agarose: 50 bp–4,000 bpDNA: 2.2% agarose: 10 bp–1,000 bpRNA: 1.2% agarose: 0.5 kb – 9.0 kbSeparation of fragments >4 kb will be improved by running longer atlower voltage.Storage:DNA: Room temperature for 5 months from dateof manufacture.RNA: Room temperature for 3 months from dateof manufacture.Shelf life may be extended <strong>with</strong> refrigerated storage.Well volume: 12+1 well: 5 µl16+1 well: 5 µl8+1 well: 12 µlGel size:70 mm (L) × 84 mm (W) × 2 mm (H)Cassette size: 115 mm (L) × 107 mm (W) × 17 mm (H)Dock size:134 mm (L) × 120 mm (W) × 54 mm (H)FlashGel Dock and CassettesNOTE: Some components and technology of the FlashGel System aresold under licensing agreements. The nucleic acid stain in this product ismanufactured and sold under license from Molecular Probes, Inc., and theFlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc,and is for use only in research applications or quality control. It is coveredby pending and issued patents. The FlashGel Dock technology containsClare Chemical <strong>Research</strong>, Inc. Dark Reader® transilluminator technologyand is covered under US Patents 6,198,107; 6,512,236; and 6,914,250.The electrophoresis technology is licensed from Temple University and iscovered under US Patent 6,905,585.Electrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com287
FlashGel SystemPower SupplySimple, Compact and PowerfulDesigned to complement the FlashGel Dock, this newpower supply boasts of simple program settings and is halfthe size of other standard power supply units. This 300 voltFlashGel Power Supply is capable of powering moststandard horizontal and vertical electrophoresis systems.■■The FlashGel Power Supply offers––Compact size––Simple easy-to-use interface––Multiple jacks to run up to two FlashGel Docks at once––Built-in timer––Easy to read digital display––Toggle between volts, current, and timePhysical SpecificationsElectrical Specifications6Terminal PairsDisplayConstruction materialUnit DimensionWeight2 Pairs3 digit LEDPolycarbonate housing and aluminum bottomplates140 × 191 × 84mm~1 kgOutput Voltage / Inc.Output Current / Inc.Max. WattRated VoltageOutput TypeControlTimerSafety Device10–300V / 1V10–400mA / 1mA60W100–240 V, 50–60 Hz, 2AConstant Voltage or Constant CurrentMicroprocessor controller1–999 minutes <strong>with</strong> alarm, continuousNo load detection; shrouded plugs and socketsElectrophoresis and Analysis / Nucleic AcidFlashGel System – Ordering InformationOrdering Information – FlashGel SystemCat. No. NA Cat. No. EU Product Name Product Description SizeFlashGel System57040 57040 FlashGel Camera Includes: Camera, hood enclosure USB cable andFlashGel Capture Software, for use <strong>with</strong> FlashGelDock57025 57025 FlashGel Dock For use <strong>with</strong> all FlashGel Cassette types each50462 50462 FlashGel Loading Dye (5X) Contains xylene cyanol 5 × 1 ml57067 57067 FlashGel System Includes: FlashGel Dock, FlashGel Camera, 9 pk eachFlashGel DNA Cassettes (1.2%, 12+1-well singletier),FlashGel Loading Dye and FlashGel DNAMarker57068 57068 FlashGel Power Supply57062 57062 FlashGel Device Pack Includes FlashGel Dock, FlashGel Power Supply,and FlashGel Camera57069 57069 FlashGel Power Supply Pack Includes FlashGel Dock and FlashGel PowerSupply57065 57065 FlashGel Camera Pack Includes FlashGel Dock and FlashGel Cameraeach288North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
FlashGel System – Ordering InformationContinuedOrdering Information – FlashGel SystemCat. No. NA Cat. No. EU Product Name Product Description SizeFlashGel System For DNA57063 57063 FlashGel DNA Kit Includes FlashGel DNA Cassettes (1.2% 12+1well single tier 9pk), FlashGel Loading Dye, andFlashGel Marker 100 bp – 4 kb57023 57023 FlashGel DNA Cassettes – 9/pk 1.2% agarose, 12+1 single-tier57029 57029 FlashGel DNA Cassettes – 9/pk 1.2% agarose, 16+1 double-tier (34-well)57031 57031 FlashGel DNA Cassettes – 9/pk 2.2% agarose, 12+1 single-tier57032 57032 FlashGel DNA Cassettes – 9/pk 2.2% agarose, 16+1 double-tier (34-well)57034 57034 FlashGel DNA Marker, 100 bp–3 kb Ready-to-load, recommended for double-tier500 µlcassettes, 100 applications50473 50473 FlashGel DNA Marker, 100 bp–4 kb Ready-to-load, recommended for 1.2% cassettes, 500 µl100 applications57033 57033 FlashGel DNA Marker, 50 bp–1.5 kb Ready-to-load, recommended for 2.2% cassettes, 500 µl100 applications57026 57026 FlashGel DNA Starter Kit Includes FlashGel Dock, FlashGel Loading Dye, eachFlashGel DNA Cassettes (1.2%, 12+1 well singletier,9 pk), FlashGel DNA Marker 100 bp–4 kb50462 50462 FlashGel Loading Dye (5X) Contains xylene cyanol 5 × 1 ml50475 50475 FlashGel QuantLadder, 100 bp(3 ng)–1.5 kb (30 ng)Ready-to-load, 50 applications 250 µlrFlashGel System for Recovery57064 57064 FlashGel Recovery Kit Includes FlashGel Recovery Cassettes 1.2% 8+1well double tier 9pk, FlashGel Recovery Buffer,FlashGel Loading Dye FlashGel QuantLadder, andVisualization Glasses57060 57060 FlashGel Recovery Buffer Ready-to-use 2 × 500 µl57022 57022 FlashGel Recovery Cassettes – 9/pk 2.2% agarose, 8+1 double-tier (18 well)57051 57051 FlashGel Recovery Cassettes – 9/pk 1.2% agarose, 8+1 double-tier (18 well)57050 57050 FlashGel Recovery Starter Kit Includes FlashGel Recovery Cassettes (1.2%, Kit8+1 well double-tier, 9 pk), FlashGel Loading Dye,FlashGel Recovery Buffer, FlashGel QuantLadder,Visualization Glasses, Control Fragment. Dock soldseparately.57061 57061 FlashGel Visualization Glasses eachFlashGel System for RNA57027 57027 FlashGel Recovery Cassettes–9/pk 1.2% agarose, 12+1 single-tier57028 57028 FlashGel Recovery Cassettes–9/pk 1.2% agarose, 16+1 double-tier (34-well)50577 50577 FlashGel RNA Marker, 0.5 kb–9 kb 50 µl57024 57024 FlashGel System for RNA Starter IIncludes FlashGel RNA Cassettes 1.2% 12+1 well KitPacksingle tier 9pk RNA Marker, Sample Buffer, andMolecular Biology Water50462 50462 FlashGel Loading Dye (5X) Contains xylene cyanol 5 × 1 ml50475 50475 FlashGel QuantLadder, 100 bp Ready-to-load, 50 applications 250 µl(3 ng)–1.5 kb (30 ng)NOTE: Due to varying storage requirements, kit components may arrive in separate shipping containers.Page 468 (detailed marker sizes)Electrophoresis and Analysis / Nucleic Acidwww.flashgel.comEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com289
Reliant MinigelsVersatile Minigels for Routine DNA Separation and RecoveryReliant Gels are versatile and convenient minigels fornearly any application. Each gel is precision manufacturedfor rapid and reproducible resolution of DNA sizes from 8 bpto 10 kb. Reliant Gels are available in a variety of wellformats and agarose concentrations, in TAE and TBE bufferand most are prestained <strong>with</strong> ethidium bromide.■■Benefits––Manufactured <strong>with</strong> high quality SeaKem® and NuSieveAgarose for reliability––Compatible <strong>with</strong> most minigel chambers––Versatile format optionsPerformance of Reliant MinigelsPanel A1X TBE + EtBrPanel B1X TAE + EtBr6Electrophoresis and Analysis / Nucleic Acid■■Applications––DNA analysis––Restriction digests––Recovery––PCR and RT-PCR––Cloning and Blotting■■Performance and Quality Tests––DNase: no activity detected––Gel performance: sharp bands and low backgroundfluorescence8-well18°C–26°C for 6–12 months depending upon agaroseconcentration6 cm24-well9.5 cm9.5 cm12-well6 cm20-well9.5 cm9.5 cm6 cmPanel A. 20 bp Ladder (1 µl), 100 bp Ladder (1 µl) and 50–1000 bp marker(2.5 µl) (all Lonza), loaded and run in a 4% NuSieve 3:1 Plus Reliant Gelcontaining ethidium bromide. Gel was run at 7 V/cm for 50 minutes using1X TBE buffer containing 0.5 µg/ml ethidium bromide.Panel B. A repeating pattern of 500 bp DNA ladder (1 µl/lane) and 1–10 kbDNA marker (2.5 µl/lane) (Lonza) run in a 1% SeaKem® Gold Reliant Gelcontaining ethidium bromide. Gel was run at 5 V/cm for 60 minutes using1X TAE buffer containing 0.5 µg/ml ethidium bromide.SpecificationsGels per box: 20Gel dimensions:6.0 cm × 9.5 cmGel thickness:5.5 mmTray dimensions:6.8 cm × 10.2 cmWell volume:
Reliant MinigelsContinuedOrdering Information – SimplyLoad LaddersCat. No. NA Cat. No. EU Product Name Product Description Range Agarose Size8-well54801 54801 Reliant Minigel TAE No stain bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 8-well (20 gels/box)54803 54803 Reliant Minigel TAE With ethidium bromide (0.5 µg/ml) bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 8-well (20 gels/box)54903 54903 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 8-well (20 gels/box)54925 54925 Reliant Minigel TAE With ethidium bromide (0.5 µg/ml) bp 20 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 8-well (20 gels/box)54927 54927 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 8-well (20 gels/box)12-well54820 54820 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 12-well (20 gels/box)54821 54821 Reliant Minigel TAE With ethidium bromide (0.5 µg/ml) bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 12-well (20 gels/box)54823 54823 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 12-well (20 gels/box)54825 54825 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 12-well (20 gels/box)20-well54907 54907 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 20-well (20 gels/box)54928 54928 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 20-well (20 gels/box)54938 54938 Reliant Minigel TBE No stain bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 20-well (20 gels/box)54939 54939 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 20-well (20 gels/box)54944 54944 Reliant Minigel TBE No stain bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 20-well (20 gels/box)24-well54813 54813 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 24-well (20 gels/box)54905 54905 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 24-well (20 gels/box)54929 54929 Reliant Minigel TBE With ethidium bromide (0.5 µg/ml) bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 24-well (20 gels/box)Contact Scientific Support to inquire about custom precast gels.Ordering Information – Supporting ProductsCat. No. NA Cat. No. EU Product Name Product Description Size54945 54945 Reliant Gel Reusable UV Transparent Tray Landscape each54946 54946 Reliant Gel Reusable UV Transparent Tray Portrait each50655 50655 DNA Loading Buffer (6X) Ficoll® based <strong>with</strong> bromophenol blue and xylene cyanol 5 × 1 ml50839 50839 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 4 l50836 50836 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 20 l50835 50835 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 10 l51216 BE51216 AccuGENE 50X TAE Buffer 2.0 M Tris-acetate, 0.05 M EDTA, pH 8.3 1 l50840 50840 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 4 l50838 50838 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 20 l50837 50837 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 10 l50843 BE50843 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 1 l50841 50841 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 4 l50844 BE50844 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 1 lElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com291
Latitude HT GelsPrecast Gels for High-throughput Separations6Latitude HT Precast Agarose Gels are large format agarosegels designed for high-throughput screening applications.These gels are precision manufactured for rapid,reproducible resolution of DNA sizes from 8 bp to 10 kb.Latitude HT Gels are available in multiple well formats(from 100–200 wells) and agarose concentrations, in TAEand TBE buffer, all prestained <strong>with</strong> ethidium bromide.■■Benefits––Manufactured <strong>with</strong> high quality SeaKem® orNuSieve Agarose for reliability––Versatile design allows you to run gels in most largesubmerged electrophoresis systems––Multichannel pipette compatible■■Applications––High-throughput DNA analysis––PCR, RT-PCR and Multiplex PCR––Genotyping––Fingerprinting––Library construction■■Performance and Quality Tests––DNase: no activity detected––Gel performance: sharp bands and low backgroundfluorescenceResolution of DNA Markers in a Latitude HT PrecastAgarose GelAlternate loads of 50–1000 bp Marker and 100 bp Ladder (Lonza) run in a2% SeaKem® LE Plus Agarose Gel in 1X TBE buffer containing 0.5 µg/mlethidium bromide. Gels run at 6 V/cm, 1 hour run using the TruBandAnchor.SpecificationsGels per box: 5Gel dimensions:24 cm × 14 cmGel thickness:6.5 mmEthidium bromide:0.5 µg/mlTray dimensions:25 cm × 15 cmWell volume:10 µl–12 µl for 50 well gels25 µl–30 µl for 25 well gelsElectrophoresis and Analysis / Nucleic Acid■■Chamber Compatibility Information––Latitude HT Gels fit most large submergedelectrophoresis systems. We recommend the followingsystems:––Owl® Centipede Horizontal System––Owl® Millipede Horizontal System––FisherBiotech® Wide Format System FB-SB-2318––Bio-Rad® Sub-Cell® Models 96 and 192––Shelton JSB-962 × 50–100-well4 × 25–100-well14 cm14 cm24 cm24 cm4 × 50–200-well14 cm18°C–26°C for 6–12 months depending upon agaroseconcentrationwww.lonza.com/sourcebook24 cm292North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Latitude HT GelsContinuedOrdering Information – Latitude HT Precast GelCat. No. NA Cat. No. EU Product Name Product Description Separation Range Agarose Size57204 57204 Latitude HT PrecastGel TAE57206 57206 Latitude HT PrecastGel TAE57224 57224 Latitude HT PrecastGel TBE57225 57225 Latitude HT PrecastGel TBE57226 57226 Latitude HT PrecastGel TBE57246 57246 Latitude HT PrecastGel TBE57255 57255 Latitude HT PrecastGel TBE57214 57214 Latitude HT PrecastGel TAE57234 57234 Latitude HT PrecastGel TBE57235 57235 Latitude HT PrecastGel TBE57236 57236 Latitude HT PrecastGel TBERelated ProductsMultichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(consecutive well), <strong>with</strong>ethidium bromide (0.5 µg/ml)Multichannel pipette compatible(consecutive well), <strong>with</strong>ethidium bromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)Multichannel pipette compatible(alternate well), <strong>with</strong> ethidiumbromide (0.5 µg/ml)bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 2 × 50-wells, 100-well(5 gels/box)bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 2 × 50-wells, 100-well(5 gels/box)bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 2 × 50-wells, 100-well(5 gels/box)bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 2 × 50-wells, 100-well(5 gels/box)bp 100 ≥ 2,000 2% SeaKem® LE Plus Agarose 2 × 50-wells, 100-well(5 gels/box)bp 100 ≥ 2,000 2% SeaKem® LE Plus Agarose 4 × 25-wells, 100-well(5 gels/box)bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 4 × 25-wells, 100-well(5 gels/box)bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 4 × 50-wells, 200-well(5 gels/box)bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 4 × 50-wells, 200-well(5 gels/box)bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 4 × 50-wells, 200-well(5 gels/box)bp 100 ≥ 2,000 2% SeaKem® LE Plus Agarose 4 × 50-wells, 200-well(5 gels/box)Contact Scientific Support to inquire about custom precast gels.Ordering Information – Supporting ProductsCat. No. NA Cat. No. EU Product Name Product Description Size56991 56991 TruBand Gel Anchor For Owl Millipede, Shelton JSB-96,Fisher SB-2318 chambers56993 56993 TruBand Gel Anchor Standard chambers50655 50655 DNA Loading Buffer (6X) Ficoll® based <strong>with</strong> bromophenol blue and xylene cyanol 5 × 1 ml50835 50835 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 10 l50836 50836 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 20 l50839 50839 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 4 l51216 BE51216 AccuGENE 50X TAE Buffer 2.0 M Tris-acetate, 0.05 M EDTA, pH 8.3 1 l50837 50837 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 10 l50838 50838 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 20 l50840 50840 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 4 l50843 BE50843 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 1 l50841 50841 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 4 l50844 BE50844 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 1 lDNA Ladders and Markers 301PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com293
Latitude MidigelsVersatile Medium-sized Precast GelsLatitude Precast Agarose Midigels are designed for highsample throughput DNA analysis applications requiringincreased resolution distance. These gels are precisionmanufactured for rapid and reproducible resolution of DNAsizes from 8 bp to 10 kb. Latitude Gels are available in avariety of well formats and agarose concentrations, in TAEand TBE buffer.■■Benefits––Manufactured <strong>with</strong> high quality SeaKem® or NuSieveAgarose for reliability––Latitude Gels fit most midigel chambers and provideoptimal performance in the Latitude Chamber■■Performance and Quality Tests––DNase: No activity detected––Gel performance: Sharp bands and low backgroundfluorescencePerformance of the 40-well Latitude Precast AgaroseMidigelsSpecificationsAlternate loads of 100 bp DNA ladder and Lonza 20 bpDNA ladder (Lonza)(1 µl marker/lane) run in a 4%NuSieve 3:1 Plus Agarose Gel in 1X TBE buffercontaining 0.5 µg/ml Ethidium Bromide. 6 V/cm,70 minute run in a 10 cm × 15 cm Latitude GelChamber using the TruBand Gel Anchor.Gels per box: 8Gel dimensions:10 cm × 15 cmGel thickness:6.0 mmEthidium bromide:0.5 µg/mlTray dimensions:10.4 cm × 15.6 cmWell volume: 10 µl–12 µl18°C–26°C for 6–12 months depending upon agaroseconcentration20-well40-well615 cm15 cmElectrophoresis and Analysis / Nucleic Acid10 cm10 cmOrdering Information – Latitude MidigelCat. No. NA Cat. No. EU Product Name Product Description Separation Range Agarose Size57200 57200 Latitude Midigel TAE With ethidium bromide (0.5 µg/ml) bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 20 wells (8 gels/box)57220 57220 Latitude Midigel TBE With ethidium bromide (0.5 µg/ml) bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 20 wells (8 gels/box)57210 57210 Latitude Midigel TAE bp 400 ≥ 10,000 1% SeaKem® LE Plus Agarose 40 wells (8 gels/box)57211 57211 Latitude Midigel TAE bp 100 ≥ 3,000 2% SeaKem® LE Plus Agarose 40 wells (8 gels/box)57230 57230 Latitude Midigel TBE bp 300 ≥ 8,000 1% SeaKem® LE Plus Agarose 40 wells (8 gels/box)57231 57231 Latitude Midigel TBE bp 100 ≥ 2,000 2% SeaKem® LE Plus Agarose 40 wells (8 gels/box)57232 57232 Latitude Midigel TBE bp 8 ≥ 1,000 4% NuSieve 3:1 Plus Agarose 40 wells (8 gels/box)Ordering Information – Supporting ProductsCat. No. NA Cat. No. EU Product Name Product Description Size56990 56990 Latitude Midigel Chamber Casting accessories not available Gel chamber56988 56988 TruBand Gel Anchor Free <strong>with</strong> your first order of Latitude Gels Latitude Chamber56989 56989 TruBand Gel Anchor Free <strong>with</strong> your first order of Latitude Gels Standard chambers294North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PAGEr Gold TBE Precast GelsPolyacrylamide Minigels for DNA SeparationPAGEr Gold TBE Precast Gels provide fine resolution of DNAfragments
Precast Gels and Related Products for RNA AnalysisClean, Reliable, Guaranteed RNase-freeReliant and Latitude Precast RNA GelsVersatile, convenient gel options for verification of RNAintegrity, Northern blotting, and analysis of RNA transcripts.Reliant and Latitude Precast RNA Agarose Gels areprecision cast in 1.25% SeaKem® Gold Agarose <strong>with</strong> MOPSbuffer and are guaranteed RNase-free. Our RNA markers,stains, and buffers are designed to optimize RNAseparations.8-well20-well■■Benefits––Guaranteed RNase free––Compatible <strong>with</strong> popular chambers■■Applications––Northern blotting––RNA integrity checks■■Performance and Quality Tests––Agarose: No RNase activity detected––Gel performance: Sharp RNA bands and low background<strong>with</strong> ethidium bromide, SYBR® Green II and GelStar®Nucleic Acid Gel Stains9.5 cm6 cmwww.lonza.com/sourcebook9.5 cmResolution of RNA Markers Run in a Reliant RNA Gel6 cm20-well40-well– 9 kb– 6 kb– 5 kb– 4 kb– 3 kb– 2.5 kb– 2 kb– 1.5 kb– 1 kb6Electrophoresis and Analysis / Nucleic Acid10 cm15 cm10 cm15 cm– 0.5 kbGel loaded <strong>with</strong> samples of RNA marker 0.5 kb –9 kb. Marker loaded at 200 ng(lanes 3 and 6) and 1 µg (lanes 1, 4, 5 and 8). Gel run at 5 V/cm for 2 hoursusing AccuGENE MOPS Buffer (1X). RNA stained for 30 minutes usingGelStar® Nucleic Acid Gel Stain (1:10,000 dilution).Ordering Information – Precast RNA GelsCat. No. NA Cat. No. EU Product Name Product Description Size54922 54922 Reliant RNA Gel System 1.25% SKG, MOPS, no stain, cassette size: 6 cm × 9.5 cm, 8-well plates 20 gels54948 54948 Reliant RNA Gel System 1.25% SKG, MOPS, no stain, 20-well plates 20 gels54975 54975 Reliant RNA Analysis Kit With formaldehyde buffer, 8 well Kit57237 57237 Latitude RNA Midigel 1.25% SKG, MOPS, cassette size: 10 cm × 15 cm, 2 × 20-well plates 8 gels57238 57238 Latitude RNA Midigel 1.25% MOPS, cassette size: 10 cm × 15 cm, 20-well plates 8 gels296North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Precast Gels and Related Products for RNA AnalysisContinuedSample BuffersReady-to-use buffers for denaturation of RNA samples forelectrophoresis on Reliant and Latitude Precast RNA Gels.Ideal for Northern blotting.Glyoxal: 18°C–26°C for 12 months; storage at 4°C willextend the stability to 2 yearsFormaldehyde: -20°C for 12 monthsOrdering Information – RNA Sample BuffersCat. No. NA Cat. No. EU Product Name Product Description Size50560 50560 Glyoxal Sample Buffer 1.7 ml50571 50571 Formaldehyde Sample Buffer RNA denaturing sample buffer, contains bromophenol blue and xylene cyanol 5 × 1 mlAccuGENE 10X MOPS BufferSpecially formulated MOPS Buffer for use <strong>with</strong> Latitude andReliant Precast Gels. Manufactured <strong>with</strong> the same reagentsused in our precast gels. Buffer contains 0.2 M MOPS (freeacid), 0.05 M sodium acetate, 0.01 M EDTA (disodium salt),and 0.01 M EGTA (free acid), pH 7.0.18°C–24°COrdering Information – AccuGENE 10X MOPS BufferCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50876 50876 AccuGENE 10X MOPS Buffer 0.2 M MOPS (free acid), 0.05 M sodium acetate, 0.01 M EDTA (disodiumsalt), 0.01 M EGTA (free acid), pH 7.0. No detectable RNase activity18°C–24°C1 lRNA Marker 0.5–9 kbRNA Markers 0.5–9 kb suitable for sizing single strandedRNA in glyoxal or formaldehyde denaturing systems. RNAmarker consists of ten RNA transcripts: 0.5, 1, 1.5, 2, 2.5, 3, 4,5, 6, and 9 kb in length. Markers can be denatured <strong>with</strong>standard procedures, and visualized on Northern blots <strong>with</strong>labeled lambda sequence. Detect 4 µg <strong>with</strong> ethidiumbromide, or smaller quantities <strong>with</strong> GelStar® or SYBR® GreenII Gel Stains.Related Products-80°C for 24 months or -20°C for 6 monthsPage 300–301 (detailed marker sizes)www.lonza.com/sourcebookOrdering Information – FlashGel RNA Marker, 0.5 kb - 9 kbCat. No. NA Cat. No. EU Product Name Product Description Size50575 50575 FlashGel RNA Marker, 0.5 kb - 9 kb RNA Marker (0.5 to 9 kb) 250 ul (50 ug) 250 µlFlashGel System for RNA 289PageElectrophoresis and Analysis / Nucleic AcidMore Precast Gels and Related Products on the next page.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com297
Precast Gels and Related Products for RNA AnalysisContinuedGelStar® Nucleic Acid Gel StainGelStar® Nucleic Acid Gel Stain is a fast-acting, fluorescentstain that is up to 15 times more sensitive than EthidiumBromide for RNA detection.––Detects 3 ng of RNA or 20 pg of dsDNAPage 302 (detailed product information)www.lonza.com/sourcebookRNA Detection <strong>with</strong> GelStar® StainA B CSamples of E. coli total RNA were denatured using thefollowing denaturants: Lane A: Formaldehyde/Formamide; Lane B: Formamide; Lane C: Glyoxal.Samples were loaded at 2 µg/lane for theformaldehyde/formamide and formamide onlydenatured samples, and 4 µg/lane for the glyoxaldenatured samples. Reliant RNA Precast AgaroseGels were run at 7 V/cm for 40 minutes in 1X MOPSBuffer and post stained <strong>with</strong> GelStar® Gel Stain andphotographed on the Clare Chemical <strong>Research</strong>, Inc.,Dark Reader® Transilluminator.Ordering Information – GelStar® Nucleic Acid Gel Stain 10,000XCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50535 50535 GelStar® Nucleic Acid Gel Stain 10,000X Supplied as a 10,000X concentrated solution in DMSO -20°C 2 × 250 µl50536 50536 SYBR® Green Gel Stain Photographic Filter Wratten® #9 18°C–26°C 3 inch squareProduct licensed from Molecular Probes, Inc.6Electrophoresis and Analysis / Nucleic AcidSYBR® Green II Nucleic Acid Gel StainSYBR® Green II Nucleic Acid Gel Stain is a highly sensitivefluorescent stain that is ideal for detection of RNA.––Detects 2 ng of RNA or 100 pg of dsDNAPage 303 (detailed product information)www.lonza.com1/sourcebookRNA Detection <strong>with</strong> SYBR® Green II StainA B CSamples of E. coli total RNA were denatured using thefollowing denaturants: Lane A: Formaldehyde/Formamide; Lane B: Formamide; Lane C: Glyoxal.Samples were loaded at 2 µg/lane for the formaldehyde/formamide and formamide only denatured samples,and 4 µg/lane f or the glyoxal denatured samples.Reliant RNA Precast Agarose Gels were run at 7 V/cm for 40minutes in 1X MOPS Buffer and post stained <strong>with</strong> SYBR®Green II Gel Stain and photographed on the Clare Chemical<strong>Research</strong>, Inc., Dark Reader® Transilluminator.Ordering Information – SYBR® Green II Nucleic Acid Gel StainCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50522 50522 SYBR® Green II Nucleic Acid Gel Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 2 × 500 µl50523 50523 SYBR® Green II Nucleic Acid Gel Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 10 × 50 µl50530 50530 SYBR® Green Gel Stain Photographic Filter Wratten® #15 18°C–26°C 3 inch squareProduct licensed from Molecular Probes, Inc.298North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Markers, Stains and BuffersOptimal Performance and ConvenienceGreat performance starts <strong>with</strong> high quality agarose andgels, but for complete assurance, you need to use highquality markers, ladders, stains, and buffers. We support abroad offering of products that complement and match theperformance of our agarose and precast gels.Rapidly estimating fragment size requires clear sharpbanding patterns on each and every gel. We offer two typesof ladders and markers: Standard and SimplyLoad. Standardmarkers and ladders are ready to dilute prior to loading yourgel, while our convenient SimplyLoad Ladders arepremixed, ready for direct loading. Our DNA quantitationladders are ideal for the accurate estimation of molecularmass of fragments from 10 ng to 100 ng.Seeing all of your data is critical to the overall success ofyour experiment. GelStar® Nucleic Acid Gel Stain clearlydetects fragments down to 20 pg of DNA. Maximize yourperformance by adding the stain directly to your gel prior tocasting or post-stain your gel. We also offer SYBR® GreenNucleic Acid Gel Stains.Finally, we offer a complete line of AccuGENEElectrophoresis and Molecular Biology Buffers to supportyour research. Our AccuGENE Buffers are formulated tooptimize performance of our agarose and precast gelproducts.Electrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com299
DNA Ladders and MarkersSizing Made Easy– 500 bp– 200 bp– 1,000 bp– 500 bp– 1,000 bp– 700 bp– 525 bp– 500 bp– 400 bp– 300 bp– 2,500 bp– 1,500 bp– 1,000 bp– 700 bp– 525 bp– 500 bp– 400 bp– 1,000 bp– 500 bp– 3,000 bp– 200 bp– 200 bp– 300 bp– 1,000 bp– 100 bp– 200 bp– 100 bp– 100 bp– 100 bp– 100 bp– 500 bp– 50 bp– 50 bp– 20 bp– 20 bp– 100 bp20 bp 20 bp Ext 50–1,000 bp 50–2,500 bp 100 bp 100 bp Ext.6Electrophoresis and Analysis / Nucleic AcidCat. No.Cat. No.Standard 50330 50320 50461 50631 50321 50322SimplyLoad 50331 50326 n/a n/a 50327 50328Ladders– 5,000 bp– 3,000 bp– 1,000 bp– 500 bp– 5,000 bp– 2,000 bp– 1,000 bp– 500 bp– 10 kb– 7 kb– 5 kb– 4 kb– 3 kb– 2.5 kb– 2 kb– 1.5 kb– 1 kb– 1,000 bp100 ng– 700 bp70 ng– 500 bp50 ng– 200 bp20 ng– 100 bp10 ng– 1,000 bp10 ng– 700 bp20 ng– 500 bp50 ng– 200 bp70 ng– 100 bp100 ngTandem 500 bp 1–10 kb QuantLadder Reverse QuantLadder RNA ladderStandard n/a 50323 50471 50334 50335 50575SimplyLoad 50333 50329 n/a 50336 50337 n/aLadders– 9 kb– 6 kb– 5 kb– 4 kb– 3 kb– 2.5 kb– 2 kb– 1.5 kb– 1 kb– 0.5 kbSimplyLoad Ladders are supplied in ready-to-load concentrations.Page 469 (detailed size information)300North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
DNA Ladders and MarkersContinuedStandard Ladders and Markers are ready-to-dilute prior toloading on your gel. Plasmid-free to ensure minimalbackground.SimplyLoad Ladders are supplied ready-to-load on your gel.No need for mixing, heating or diluting prior to loading. Plasmidfreeto ensure minimal background.Standard Ladders and Markers: 4°C or -20°CSimplyLoad Ladders: 4°COrdering Information – Standard Ladders and MarkersCat. No. NA Cat. No. EU Product Name Product Description Range Applications SizeStandard Ladders50320 50320 20 bp Extended Range DNA Ladder 20 bp - 1,000 bp 100 150 µl50321 50321 100 bp DNA Ladder Concentration: 100 ng/μl 100 bp - 1,000 bp 100 160 µl50322 50322 100 bp Extended Range DNA Ladder 100 bp - 3,000 bp 100 150 µl50323 50323 500 bp DNA Ladder 500 bp - 8,000 bp 200 300 µl50330 50330 20 bp DNA Ladder Standard Ladder 20 bp - 500 bp 100 150 µlStandard Quantitation Ladders50334 50334 DNA QuantLadder 100 bp - 1,000 bp 50 125 µl50335 50335 DNA Reverse QuantLadder 100 bp - 1,000 bp 50 125 µlStandard DNA Ladders50461 50461 50 bp DNA Marker 50 bp - 1,000 bp 50 250 µl50471 50471 1kb DNA Marker 1 kb - 10 kb 100 2 × 250 µl50631 50631 50 bp DNA Marker 50 bp to 2,500 bp 50 250 µlOrdering Information – SimplyLoad LaddersCat. No. NA Cat. No. EU Product Name Product Description Range Applications SizeSimplyLoad DNA Ladders50326 50326 SimplyLoad 20 bp Extended Range DNA Ladder 20 bp - 1,000 bp 100 500 µl50327 50327 SimplyLoad 100 bp DNA Ladder 100 bp - 1,000 bp 100 500 µl50328 50328 SimplyLoad 100 bp Extended Range DNA Ladder 100 bp - 3,000 bp 100 500 µl50329 50329 SimplyLoad 500 bp DNA Ladder 500 bp - 8,000 bp 100 500 µl50331 50331 SimplyLoad 20 bp DNA Ladder 20 bp - 500 bp 100 500 µl50333 50333 SimplyLoad Tandem DNA Ladder 100 bp - 12,000 bp 100 500 µlSimplyLoad Quantitation Ladders50336 50336 SimplyLoad DNA QuantLadder SimplyLoad DNA Quant 100 bp - 1,000 bp 50 250 µl50 applications50337 50337 SimplyLoad DNA Reverse QuantLadder SimplyLoad DNAReverse 50 applications100 bp - 1,000 bp 50 250 µlRelated ProductsAgarose 269Precast Gels 282PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com301
GelStar® Nucleic Acid Gel StainExquisitely Sensitive In-gel Stain for DNA and RNAGelStar® Nucleic Acid Gel Stain is a highly sensitivefluorescent stain for detecting both DNA and RNA. AddGelStar® Stain to your agarose solution prior to casting, orpost-stain your gels. GelStar® Stain exhibits exceptionalsignal-to-noise ratio <strong>with</strong> minimal background.■■Benefits––Maximum sensitivity – Detect as little as 20 pg ofdsDNA or 3 ng of RNA––Versatile – Use for agarose or polyacrylamide gelelectrophoresis, ideal alternative to silver staining––Ultimate user flexibility – Add GelStar® Stain prior to gelcasting or post-stain, no destaining required––Complete staining solution for all types of nucleic acids––Detect fragments <strong>with</strong> either a standard 300 nm UVtransilluminator or the Clare Chemical <strong>Research</strong>, Inc.,Dark Reader® Transilluminator■■Applications––DNA and RNA detection––SSCP and heteroduplex analysis-20° C for stain 18°C–26°C for photographic filterGelStar® Stain Versus Ethidium BromideGelStar® Stain1 2 3 4 5 6 7 8Ethidium Bromide1 2 3 4 5 6 7 8Serial dilution of SimplyLoad DNA QuantLadder on 2% Reliant PrecastGels post-stained <strong>with</strong> 1X GelStar® Stain (top) or 0.5 μg/ml ethidiumbromide (bottom) for 45 minutes.6Electrophoresis and Analysis / Nucleic Acid302www.lonza.com/sourcebookStain and Method ssDNA dsDNAGelStar® Stain – in gel 25 pg 20 pgEthidium bromide, no destain 1.25 ng 350 pgEthidium bromide, destain 350 pg 100 pgSYBR® Green I or II Stain 60 pg 20–30 pgThe FlashGel System includes gel cassettes prestained<strong>with</strong> a similar high-sensitivity stain. Refer to page 283–289GelStar® Gel Stain Photographic Filter––Use for optimal sensitivity <strong>with</strong> black and white film––Suitable for use <strong>with</strong> most Polaroid® Documentation orCamera SystemsRelated ProductsGelStar® StainIn-Gel Post-StainedLonza’s 500 bp DNA Ladder wasseparated on 1% SeaKem® GTGAgarose gels 20 cm long, 4 mm thick,run in 1X TBE buffer (Prepared fromLonza’s AccuGENE 10X TBE Buffer) at6 V/cm for 3 hours. GelStar® Stain wasdiluted 1:10,000 and added directly tothe agarose or the gel was post stainedfor 30 minutes in a 1 : 10,000 dilutionof GelStar® Stain in buffer. Lane 1 : 10ng DNA/band; Lane 2 : 5 ng DNA/band;Lane 3: 2.5 ng DNA/band; Lane 4 : 1.25ng DNA/band.Ordering Information – GelStar® Nucleic Acid Gel Stain 10,000XCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50535 50535 GelStar® Nucleic Acid Gel Stain 10,000X Supplied as a 10,000X concentrated solution in DMSO -20°C 2 × 250 µl50536 50536 SYBR® Green Gel Stain Photographic Filter Wratten® #9 18°C–26°C 3 inch squareProduct licensed from Molecular Probes, Inc.Agarose 269DNA Ladders 301North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.comPage
SYBR® Green Nucleic Acid Gel StainsSensitive Fluorescent Stains for DNA and RNASYBR® Green Nucleic Acid Gel Stains are fluorescent stainsfor detecting DNA and RNA, exhibiting excellent signal-tonoiseratio <strong>with</strong> minimal background. SYBR® Green Stainsare more sensitive than standard stains, making themconvenient alternatives to silver staining and radioisotopes.For maximum detection, gels should be post-stained andphotographed <strong>with</strong> the SYBR® Green Photographic Filter.SYBR® Green I Stain––Detects as little as 60 pg of dsDNA and 1 ngoligonucleotides––Optimal for analysis of PCR products in gels, apoptosisstudies, and heteroduplex analysisSYBR® Green II Stain––Detects 100 pg of ssDNA and 2 ng of RNA––Optimal for RNA gel electrophoresis and SSCP analysisRNA Detection <strong>with</strong> SYBR® Green II StainA B CSYBR® Green I Stain200 100 50 10 5.0 1.0Samples of E. coli total RNA were denatured usingthe following denaturants: Lane A: Formaldehyde/Formamide; Lane B: Formamide; Lane C: Glyoxal.Samples were loaded at 2 µg/lane for theformaldehyde/formamide and formamide onlydenatured samples, and 4 µg/lane for the glyoxaldenatured samples. Reliant RNA Precast AgaroseGels were run at 7 V/cm for 40 minutes in 1X MOPSBuffer and post stained <strong>with</strong> SYBR® Green II Gel Stainand photographed on the Clare Chemical <strong>Research</strong>,Inc., Dark Reader® Transilluminator.DNA Stained <strong>with</strong> SYBR® Green I Stain or EthidiumBromideEthidium Bromide Stain200 100 50 10 5.0 1.0 ng DNASYBR® Green Gel Stain Photographic Filter––Required for optimal sensitivity <strong>with</strong> black and white film––Suitable for use <strong>with</strong> most Polaroid® Systems■■Applications––DNA and RNA detection––SSCP and heteroduplex analysis-20° C for stain18°C–26°C for photographic filterwww.lonza.com/sourcebookRelated ProductsDNA samples (pBR322 Msp I digest) ranging from 1 to 200 ng per lanewere separated on a 10 cm × 16 cm × 0.1 cm, 4% vertical MetaPhorAgarose gel prepared in 1X TBE buffer. The gel was run for 1 hour at 488 V/cm.Following electrophoresis the gel was divided into two, and one half wasstained <strong>with</strong> 1 µg/ml ethidium bromide while the other was stained <strong>with</strong>SYBR® Green I Stain (1:10,000 dilution of stock). Detection was achieved<strong>with</strong> standard 300 nm UV transillumination.Ordering Information – SYBR® Green I Nucleic Acid StainCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50513 50513 SYBR® Green I Nucleic Acid Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 10 × 50 µl50512 50512 SYBR® Green I Nucleic Acid Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 2 × 500 µl50523 50523 SYBR® Green II Nucleic Acid Gel Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 10 × 50 µl50522 50522 SYBR® Green II Nucleic Acid Gel Stain Supplied as a 10,000X concentrated solution in DMSO -20°C 2 × 500 µl50530 50530 SYBR® Green Gel Stain Photographic Filter Wratten® #15 18°C–26°C 3 inch squareProduct licensed from Molecular Probes, Inc.AccuGENE Buffers 304Agarose 269Precast Gels 282PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com303
AccuGENE Molecular Biology BuffersConvenient and Ready-to-useAccuGENE Molecular Biology Buffers are ready-to-usesolutions ideal for a wide range of molecular biologyapplications.18°C–24°Cwww.lonza.com /sourcebook■■Benefits––Reliable – Manufactured according to strict qualitycontrol standards to ensure lot-to-lot consistency––High quality – Guaranteed DNase, RNase, and proteasefree––Efficient – Ready-made solutions eliminate experimentpreparation time––Flexible – Customized solutions are available to meetindividual needs6Electrophoresis and Analysis / Nucleic AcidOrdering Information – AccuGENE BuffersCat. No. NA Cat. No. EU Product Name Product Description Size51200 BE51200 AccuGENE Molecular Biology Water 1 l51223 51223 AccuGENE Molecular Biology Water 10 l51224 51224 AccuGENE Molecular Biology Water 20 l51201 51201 AccuGENE 0.5 M EDTA Solution Disodium salt, pH 8.0 100 ml51234 51234 AccuGENE 0.5 M EDTA Solution Disodium salt, pH 8.0 1 l51202 51202 AccuGENE 5 M Sodium Chloride 1 l51206 51206 AccuGENE 10% SDS Monosodium salt 100 ml51213 51213 AccuGENE 10% SDS Monosodium salt 500 ml51203 51203 AccuGENE 3 M Sodium Acetate pH 5.2 500 ml51205 BE51205 AccuGENE 20X SSC Buffer 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0 1 l51233 BE51233 AccuGENE 20X SSC Buffer 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0 10 l51232 BE51232 AccuGENE 20X SSC Buffer 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0 20 l51214 BE51214 AccuGENE 20X SSPE Buffer 3.0 M NaCl, 0.2 M NaH2PO4, H2O, 0.02 M EDTA, pH 7.4 1 l51246 51246 AccuGENE 20X SSPE Buffer 3.0 M NaCl, 0.2 M NaH2PO4, H2O, 0.02 M EDTA, pH 7.4 10 l51235 51235 AccuGENE 1X TE Buffer 0.01 M Tris, 0.001 M EDTA (disodium salt), pH 7.4 500 ml51242 51242 AccuGENE 1X TE Buffer 0.01 M Tris, 0.001 M EDTA (disodium salt), pH 7.4 10 l51236 51236 AccuGENE 1 M Tris HCl Buffer pH 7.2 1 l51237 51237 AccuGENE 1 M Tris HCl Buffer pH 7.4 1 l51238 51238 AccuGENE 1 M Tris HCl Buffer pH 8.0 1 l51217 51217 AccuGENE LB Broth (Luria Bertani Medium) 10 g/l Bacto-Tryptone, 5 g/l Bacto-Yeast Extract, and 10 g/l NaCl 500 ml51219 51219 AccuGENE Super Broth (Terrific Broth) 12 g/l Bacto-Tryptone, 24 g/l Bacto-Yeast Extract, 6.3 g/l glycerol, 2.5 g/l 500 mlK2HPO4, and 3.8 g/l KH2PO4, pH 7.251225 51225 AccuGENE 1X PBS 1.7 mM KH2PO4, 5 mM Na2HPO4, 150 mM NaCl, pH 7.4 1 l51226 51226 AccuGENE 10X PBS 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7.4 1 l51229 51229 AccuGENE Neutralization Solution 1.5 M NaCl, 1.0 M Tris, pH 7.5 1 l51230 BE51230 AccuGENE Neutralization Solution 1.5 M NaCl, 1.0 M Tris, pH 7.5 10 l304North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
AccuGENE Electrophoresis BuffersOptimal PerformanceAccuGENE Electrophoresis Buffers are formulated formaximum performance and convenience, and are optimizedfor use <strong>with</strong> our agarose and precast gels.AccuGENE Buffers for DNA, RNA, and protein electrophoresisare prepared <strong>with</strong> high quality reagents and use18 megOhm water. Products are filtered using a 0.2-micronfilter, and are guaranteed DNase/RNase free.■■Benefits––Reliable – Manufactured according to strict qualitycontrol standards to ensure lot-to-lot consistency––Efficient – Ready-to-use solutions eliminate experimentpreparation time––Flexible – Customized solutions are available to meetindividual needs18°C–24°C, 4°C for CE BufferOrdering Information – AccuGENE BuffersCat. No. NA Cat. No. EU Product Name Product Description SizeBuffers for DNA Electrophoresis50844 BE50844 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 1 l50841 50841 AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), pH 8.0 4 l51216 BE51216 AccuGENE 50X TAE Buffer 2.0 M Tris-acetate, 0.05 M EDTA, pH 8.3 1 l50839 50839 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 4 l50835 50835 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 10 l50836 50836 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), pH 8.3 20 l50843 BE50843 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 1 l50840 50840 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 4 l50837 50837 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 10 l50838 50838 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), pH 8.3 20 lBuffers for Capillary DNA Sequencing50864 50864 AccuGENE 10X CE Buffer Formulated for use on the ABI Prism® 3700 DNA Analyzer 4 l50873 50873 AccuGENE 10X CE Buffer Formulated for use on the ABI Prism® 3100 Genetic Analyzer 25 mlBuffers for RNA Electrophoresis50876 50876 AccuGENE 10X MOPS Buffer 0.2 M MOPS (free acid), 0.05 M sodium acetate, 0.01 M EDTA (disodium salt), 1 l0.01 M EGTA (free acid), pH 7.0. No detectable RNase activityElectrophoresis Loading Buffers50655 50655 DNA Loading Buffer (6X) Ficoll® based <strong>with</strong> bromophenol blue and xylene cyanol 5 × 1 ml50632 50632 Triple-Dye Loading Buffer (6X) Contains bromophenol blue, xylene cyanol, and orange G 1.1 mlBuffers for Protein Electrophoresis50879 BE50879 AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine 1 l50881 50881 AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine 4 l50882 50882 AccuGENE 10X Tris-Glycine SDS Buffer 0.25 M Tris base, 1.92 M Glycine, 1% SDS 4 lRelated ProductsPageAgarose 269Precast Gels 282Protein Electrophoresis Products 310Electrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com305
GelBond® FilmAgarose Support FilmGelBond® Film is a transparent, flexible polyester filmdesigned to support agarose gels. Gels cast on GelBond®Film remain permanently attached to the film throughelectrophoresis or immunodiffusion and all subsequentfixing, staining, destaining, and drying procedures (gelsremain flexible after drying). GelBond® Film is availableeither as precut sheets or rolls.■■Applications––Drying and support of agarose gels18°C–26°C■■Benefits––Reliable – Agarose gels cast on GelBond® Film retaintheir original dimensions during staining and afterdrying––Durable – Gels, particularly thin ones, are easier tohandle during staining, destaining, and drying whensupported––Convenient – Gel orientation can be recorded directlyon the GelBond® Film prior to castingNOTE: Polyester films will not transmit light of less than310 nm, and will fluoresce at higher wavelengths.6Electrophoresis and Analysis / Nucleic AcidOrdering Information – GelBond® Film Sheets and RollsCat. No. NA Cat. No. EU Product Name Product Description Sheet Size (mm) Chamber Compatibility53734 53734 GelBond® Film Sheets For agarose gels, 0.2 mm thick 85 mm × 100 mm (100 sheets)53745 53745 GelBond® Film Sheets For agarose gels, 0.2 mm thick 110 mm × 125 mm (100 sheets)53746 53746 GelBond® Film Sheets For agarose gels, 0.2 mm thick 100 mm × 150 mm (100 sheets) Bio-Rad® Wide Mini-Sub® Cell, Bio-Rad® Sub-Cell® (H)53748 53748 GelBond® Film Sheets For agarose gels, 0.2 mm thick 110 mm × 205 mm (100 sheets)53749 53749 GelBond® Film Sheets For agarose gels, 0.2 mm thick 160 mm × 180 mm (100 sheets) Hoefer® SE400, Hoefer® SE600 (V),Bio-Rad PROTEAN® II xi (V)53759 53759 GelBond® Film Sheets For agarose gels, 0.2 mm thick 125 mm × 245 mm (100 sheets)53761 53761 GelBond® Film Sheets For agarose gels, 0.2 mm thick 124 mm × 258 mm (100 sheets) GE Multiphor® (H)53740 53740 GelBond® Film Rolls For agarose gels, 0.2 mm thick 102 mm × 16.5 m (roll)rolls, 16.5 meters long53750 53750 GelBond® Film Rolls For agarose gels, 0.2 mm thick 102 mm × 16.5 m (roll)rolls, 16.5 meters long53780 53780 GelBond® Film Rolls For agarose gels, 0.2 mm thick 203 mm × 16.5 m (roll)rolls, 16.5 meters longCustom-cut GelBond® Film is available upon special request. Please inquire for pricing and availability.(H) = Horizontal; (V) = VerticalRelated ProductsAgarose 269Page306North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
GelBond® PAG FilmPolyacrylamide Support FilmGelBond® PAG Film is a transparent, flexible polyester filmdesigned to support polyacrylamide or MDE Gels. Theacrylamide monomers covalently attach to the coating onthe film during the polymerization reaction. Gels remainpermanently attached to the film through electrophoresisand all subsequent fixing, staining, destaining, and dryingprocedures.■■Benefits––Reliable – Polyacrylamide gels retain their originaldimensions during staining and after drying––Durable – Gels, particularly thin ones, are easier tohandle during staining, destaining, and drying whensupported––Convenient – Gel orientation can be recorded directlyon the GelBond® PAG Film prior to castingNOTE: Polyester films will not transmit light of less than310 nm, and will fluoresce at higher wavelengths.■■Applications––Drying and support of polyacrylamide gels18°C–26°C, protect from lightOrdering Information – GelBond® Film Sheets and RollsCat. No. NA Cat. No. EU Product Name Product Description Sheet Size (mm) Chamber Compatibility54711 54711 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 138 mm × 158 mm (50 sheets)54723 54723 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 160 mm × 180 mm (50 sheets) Hoefer® SE400, SE600,Bio-Rad® PROTEAN® II54727 54727 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 124 mm × 258 mm (50 sheets) GE Multiphor®54729 54729 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 220 mm × 165 mm (50 sheets)54731 54731 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 199 mm × 264 mm (50 sheets)54733 54733 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 203 mm × 260 mm (50 sheets) GE Multiphor® ll54735 54735 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 195 mm × 370 mm (50 sheets) Biometra® SA-3254746 54746 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 350 mm × 430 mm (10 sheets) X-ray sizeCustom-cut GelBond® PAG Support Film is available upon special request. Please inquire for pricing and availability.Related ProductsProSieve 50 Acrylamide Gel Solution Gels 329PageElectrophoresis and Analysis / Nucleic AcidEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com307
GelBond® PAG Support FilmPolyacrylamide Support FilmGelBond® PAG Support Film is a transparent, flexiblepolyester film designed to support polyacrylamide Gels. Theacrylamide monomers covalently attach to the coating onthe film during the polymerization reaction. Gels remainpermanently attached to the film through electrophoresisand all subsequent fixing, staining, destaining, and dryingprocedures.■■Benefits––Reliable – Polyacrylamide gels retain their originaldimensions during staining and after drying––Durable – Gels, particularly thin ones, are easier tohandle during staining, destaining, and drying whensupported––Convenient – Gel orientation can be recorded directlyon the GelBond® PAG Support Film prior to castingNOTE: Polyester films will not transmit light of less than 310 nm, andwill fluoresce at higher wavelengths.GelBond® PAG Support Film■■Applications––Drying and support of polyacrylamide gels18°C–26°C, protect from light6Electrophoresis and Analysis / Nucleic AcidOrdering Information – GelBond® Film Sheets and RollsCat. No. NA Cat. No. EU Product Name Product Description Sheet Size (mm) Chamber Compatibility54711 54711 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 138 mm × 158 mm(50 sheets)54723 54723 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 160 mm × 180 mm(50 sheets)54727 54727 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 124 mm × 258 mm(50 sheets)54729 54729 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 220 mm × 165 mm(50 sheets)54731 54731 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 199 mm × 264 mm(50 sheets)54733 54733 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 203 mm × 260 mm(50 sheets)54735 54735 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 195 mm × 370 mm(50 sheets)54746 54746 GelBond® PAG Support Film Sheets For polyacrylamide gels, 0.2 mm thick 350 mm × 430 mm(10 sheets)Related ProductsHoefer® SE400, SE600,Bio-Rad® PROTEAN® IIGE Multiphor®GE Multiphor® llBiometra® SA-32X-ray sizeCustom-cut GelBond® PAG Support Film is available on special request. Please inquire for pricing and availability.AccuGENE Buffers 304ProSieve Protein Marker 319SYPRO® Protein Stains 322Page308North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Protein Electrophoresis and AnalysisHigh-Performance Products that are Fast and Easy to UseProtein Electrophoresis and AnalysisIntroduction310Precast GelsNew! PAGEr EX Protein Trial Kits 311PAGEr EX Gels 312ProSieveEX Stains 313Prosieve EX Running and Transfer Buffers 314PAGEr Gold Precast Gels 315Selecting the Best PAGEr Gold Precast Gel 316PAGEr Gold Scouting Kit 316PAGEr Minigel Chamber 317ProSieve Color Protein Markers 318ProSieve Protein Markers 319ProSieve ProTrack Dual Color Protein Loading Buffer 319AccuGENE Protein Electrophoresis Buffers 320ProSieve Blue Protein Staining Solution 320SYPRO® Protein Gel Stains 321SYPRO® Ruby Protein Gel Stain 322SYPRO® Red Protein Gel Stain 322SYPRO® Tangerine Protein Gel Stain 323SYPRO® Ruby Protein Blot Stain 323SYPRO® Protein Gel Stain Photographic Filter 324IsoGel Agarose and Precast IsoGel Agarose IEF Plates325IsoGel Agarose 325Precast IsoGel Agarose IEF Plates 326Agarose for Protein Separation 327Agarose for Protein Separation 328ProSieve 50 Acrylamide Gel Solution 3296Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com309
IntroductionFaster Protein Solution for Separations, Blotting and Staining6Lonza’s new protein solution is addressing the need for afaster, more efficient and reliable process <strong>with</strong> proteinelectrophoresis. Ultimately, these new products combined,take protein separation, western and transfer blotting, andstaining from over 5 hours down to less than 1 hour.Our new PAGEr EX Protein Staining Kits and PAGEr EXProtein Transfer/Western Blot Kits are demo kits designedto combine the total solution for the ultimate fast separation<strong>with</strong> staining and transfer in less than 30 minutes.Each individual component offers a unique solution and canbe incorporated into your current protein process:––PAGEr EX Gels were designed for fast 15 minuteseparation, ambient shipping, and are run <strong>with</strong> usingProSieve EX Running Buffer.––ProSieve EX Safe Stain takes your staining processdown to just 1 step in 10 minutes.––ProSieve EX Stain Enhancer is combined <strong>with</strong>Commassie for a cost effective, 15 minute staining.––ProSieve EX Western Blot Transfer Buffer can be used<strong>with</strong> any Tris-Glycine gels for a 10 minute transfer.––ProSieve EX Running Buffer offers a 10–20 minuteseparation time for any Tris-glycine gels.––ProSieve QuadColor Protein Marker provides accurateconfirmation of protein transfer in the range of4.6kDa–300kDa.Lonza Protein Solution Time SavingsMinutes1009080706050403020100PrepRunBlotStainManual gel processLonza Fast Protein ProductsThe time savings and convenience can help your research <strong>with</strong> each stageof the protein process, from prep to stain or blot time.Electrophoresis and Analysis / Protein310North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
New! PAGEr EX Protein Trial KitsComplete Solution in Less than 1 HourThe new staining and blotting kits are designed forconvenience <strong>with</strong> everything you need to improve andsimplify your protein electrophoresis process.■■Kits consist of:––PAGEr EX Protein Transfer/Western Blotting Kit –2 PAGEr EX Gels, ProSieve EX Running Buffer,ProSieve EX Western Blot Transfer Buffer and aProSieve Quad Color Marker––PAGEr EX Protein Staining Kit – 2 PAGEr EX Gels,ProSieve EX Running Buffer, ProSieve EX Safe Stainand a ProSieve Quad Color MarkerOrdering Information – PAGEr Protein Trial KitsCat. No. NA Cat. No. EU Product Name Product Description Range201741 201741 Fast Protein Transfer Blotting Kit Mid/high, cassette size: 10 cm × 10 cm, 12well 25–250 kDa201742 201742 Fast Protein Transfer Blotting Kit Mid/high, cassette size: 10 cm × 10 cm, 12well 25–250 kDa201743 201743 Fast Protein Transfer Blotting Kit Low/mid, cassette size: 9 cm × 10 cm, 12well 25–200 kDa201744 201744 Fast Protein Transfer Blotting Kit Low/mid, cassette size: 10 cm × 10 cm, 12well 25–200 kDa201745 201745 Fast Protein Staining Kit Low/mid, cassette size: 9 cm × 10 cm, 12well 25–200 kDa201746 201746 Fast Protein Staining Kit Low/mid, cassette size: 10 cm × 10 cm, 12well 25–200 kDa201747 201747 Fast Protein Staining Kit Mid/high, cassette size: 9 cm × 10 cm, 12well 25–250 kDa201748 201748 Fast Protein Staining Kit Mid/high, cassette size: 10 cm × 10 cm, 12well 25–250 kDa6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com311
PAGEr EX GelsRedesigned for Speed and Longer Shelf LifePAGEr EX Gels are completely redesigned formulations<strong>with</strong> faster run times and longer shelf life. They cover the fullprotein size range <strong>with</strong> fewer configurations making iteasier to choose the best one for your needs. They are alsocompatible <strong>with</strong> a wide range of chambers. These are morethan just another type of protein gel, they are a solution for:■■Benefits––Fast separation, 20–25 minutes used <strong>with</strong> ProSieve EXRunning Buffer––Reduce your costs <strong>with</strong> ambient shipping––12 month shelf life■■Performance and Quality Tests––Every lot of PAGEr EX Gels is functionally tested and100% guaranteed2°–8°C6PAGEr EX Gels Performance and SpecificationsWell formats Size Separation Equivalent concentration Cassette Dimensions Buffer needed Chambers Types12 well, 16 well Low/Med range: 5–225 kDAMed/High range: 10–350 kDA10%4–12%9x10cm, 10x10cmProSieve EX RunningBufferSee chamber compatibilityElectrophoresis and Analysis / ProteinOrdering Information – PAGEr EX GelsCat. No. NA Cat. No. EU Product Name Product Description Range12-well58722 58722 PAGEr EX Gels Mid/high, cassette size: 9 cm × 10 cm, 12 well 10–350 kDa59722 59722 PAGEr EX Gels Mid/high, cassette size: 10 cm × 10 cm, 12 well 10–350 kDa58702 58702 PAGEr EX Gels Low/mid, cassette size: 9 cm × 10 cm, 12 well 5–225 kDa59702 59702 PAGEr EX Gels Low/mid, cassette size: 10 cm × 10 cm, 12 well 5–225 kDa16-well58724 58724 PAGEr EX Gels Mid/high, cassette size: 9 cm × 10 cm, 16 well 10–350 kDa59724 59724 PAGEr EX Gels Mid/high, cassette size: 10 cm × 10 cm, 16 well 10–350 kDa58714 58714 PAGEr EX Gels Low/mid, cassette size: 9 cm × 10 cm, 16 well 5–225 kDa59714 59714 PAGEr EX Gels Low/mid, cassette size: 10 cm × 10 cm, 16 well 5–225 kDa312North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ProSieveEX StainsRevolutionary, Fast and SafeThese new staining products are revolutionary for increasedspeed and process improvements. With special featuresthat make each product unique, choosing the right stain foryour research is easy.ProSieve EX Stain Enhancer––ProSieve EX Safe Stain, ultimate fast solution that canprovide a one step, safe stain in 10 minutes.––ProSieve EX Stain Enhancer offers a cost effectiveoption for Coomassie stain users simply in 15 minutes.ProSieve EX Safe Stain <strong>with</strong> PAGEr EX Gels OffersBetter Results in Half the TimePAGEr EX Gels. stained using ProSieve EX Stain Enhancer and 0.0125%Coomassie Brilliant Blue G in water: 15 min gel run <strong>with</strong> ProSieve EXRunning Buffer at 275V, 10 minutes ProSieve EX Stain Enhancer, 5minutes Coomassie brilliant Blue (total 30 minutes)15 min gel run <strong>with</strong> PAGEr EX Gels and ProSieve EX Running Buffer at 275V,10 minutes ProSieve EX Safe Stain , (total 25 minutes)62°–8°COrdering Information – ProSieveEX StainsCat. No. NA Cat. No. EU Product Name Product Description Size201455 201455 ProSieve EX Safe Stain One step, ten minute protein stain that is non-toxic 1 l201456 201456 ProSieve EX Safe Stain One step, ten minute protein stain that is non-toxic 25 ml201369 201369 ProSieve EX Stain Enhancer Two step, fifteen minute protein stain enhancing1 lCoomassie Blue201370 201370 ProSieve EX Stain Enhancer Two step, fifteen minute protein stain enhancingCoomassie Blue25 mlElectrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com313
Prosieve EX Running and Transfer BuffersNew protein separation and western blot transfer buffersare modified formulations that perform just like tris-glycine,but significantly accelerate run and transfer times <strong>with</strong>outcompromising results. Tris-glycine SDS buffers have beenrecognized as the gold standard for analyzing proteins byPAGE, for decades. Now, the standard 2-hour method forprotein separation and transfer can be reduced to less than30 minutes <strong>with</strong> these buffers:ProSieve EX Transfer/Western Blot Buffer <strong>with</strong> PAGErEX Gels Provides Protein Confirmation in Minutes■■Benefits––Separation in 10–20 minutes––Transfer in 10 minutes––Compatibility <strong>with</strong> standard gel systems and protocols––Razor sharp resolution2°–8°CProSieve EX Transfer/Western Blot Buffer run <strong>with</strong> PAGEr EX Gels andProSieve Running Buffer transferred to PVDF6Electrophoresis and Analysis / ProteinOrdering Information – ProSieve EX BuffersCat. No. NA Cat. No. EU Product Name Product Description Size200309 200309 ProSieve EX Transfer Buffer Ten minute protein transfer buffer 1 l200307 200307 ProSieve EX Running Buffer Less than 30 minute protein running buffer 1 l200308 200308 ProSieve EX Running Buffer Less than 30 minute protein running buffer 4 lRelated ProductsPAGEr EX Gels 312ProSieve QuadColor Protein Marker 318ProSieve EX Safe Stain 313ProSieve Stain Enhancer 313Page314North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PAGEr Gold Precast GelsReliable, Easy-to-use MinigelsFresh GelsEvery TimePAGEr Precast Gels are easy-to-use protein minigels thatoffer sharper resolution, more consistent protein transfer,and longer shelf life than any other Tris-Glycine gel. PAGErGels are easy-to-use and compatible <strong>with</strong> most minigelchambers.■■Benefits––Razor sharp resolution – Crisp separation of proteins5 kDa–300 kDa––Easy-to-use – Marked sample lanes for easy loadingand simple twist open design––Compatible – Two sizes to fit most chambers––Versatile – Multiple well formats and gel concentrations––Tris-Glycine buffer – Traditional Laemmli separation––Fresh – We ship fresh gels every time for guaranteedperformance■■Applications––Western blotting––Denaturing and native protein electrophoresis––2D electrophoresis■■Performance and Quality Tests––Every lot of PAGEr Precast Gels is functionally testedand 100% guaranteedWe offer over 70 format options for denatured and nativeprotein separation over a wide molecular weight range, in anarray of configurations in both 9 cm × 10 cm and 10 cm × 10cm sizes to fit popular chambers. See chamber compatibilitychart (at right) to determine theright gel size for your system.■■Chamber Compatibility––PAGEr Precast Gels are available in 9 cm × 10 cm and10 cm × 10 cm sizes and fit most standard mini-verticalsystems––Some chambers may require modifications for optimalfit <strong>with</strong> PAGEr Precast GelsSpecificationsCassetteDimensionsCassetteThicknessGelDimensions9 cm × 10 cm (L × W) 0.49 cm 7.1 × 8.3 (L × W) × 0.1 cm10 cm × 10 cm (L × W) 0.55 cm 8.1 × 8.3 (L × W) × 0.1 cmGel matrix/bufferPolyacrylamide/Tris-GlycineNOTE: Gels do not contain SDS. Add SDS tosample buffer to create denaturing runningconditions.Stacking gel4% stacking gelWell formats2D well, 8+1 well*, 10-well, 12-well, 16-well,17-well*CassettesPlasticStorage/shelf life2°C–8°C for 3.5 months from date ofmanufactureGuaranteed 10 weeks shelf life upon receipt*multichannel pipette compatible well formats■■PAGEr Precast Gel comb formats––Comb configurations are designed for a range of samplevolumes and through put, including multichannelpipette compatible formatsNumber of wells: 10-well 12-wellRecommended load 32 µl 20 µlvolume:Number of wells: 16-well 17-well*Recommended load 14 µl 14 µlvolume:Number of wells: 2D-well 8 + 1-well*Recommended loadvolume:Standard Vertical SystemsPAGEr Minigel Chamber550 µl sample, or 7 cmIPG strip, 12 µl markerBio-Rad® Mini-PROTEAN® II, Mini-PROTEAN® 3 ,Mini-PROTEAN® Tetra, Mini-PROTEAN® Dodeca andReady Gel® Cell Systems.Reverse the inner coregasket so the flat side faces outward.Novex® XCell SureLock® Mini-Cell or XCell IIRequest the spacer for the XCell SureLock® Mini-Cell Chamber from Scientific Support, (Cat. No.59900).FisherBiotech® Vertical Minigel FBVE121,Owl Separations Systems Wolverine P82Chamber comes <strong>with</strong> 2 sets of wedges. Use thethinner wedges for the PAGEr Gold Gels.FisherBiotech® Vertical Minigel FB-VE101,Owl Separations Systems Penguin Model P8DSRequest adaptor for these chambers from ScientificSupport, (Cat. No. 59902).Hoefer® Mighty Small (SE250, SE260)If using SE250 replace the buffer chamber <strong>with</strong> a’Deep lower buffer chamber for the SE260’, ordernumber 80–6148–78, from GE Healthcare.Daiichi 2, ISS chambersHoefer® Mighty Small (SE260)EC 120 Mini Vertical Gel SystemBiometra® Mini V ChamberCBS Scientific MGV System,(10 cm × 8 cm units)Sigma-Aldrich Mini Techware(11.3 cm × 10 cm units)Zaxis System 2000Hoefer® Mini VE30 µl sample 12 µlmarker*Multichannel pipette compatiblePAGEr Gels9 cm × 10 cm or10 cm × 10 cm gels*9 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels*10 cm × 10 cm gels10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels*10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels9 cm × 10 cm gels9 cm × 10 cm gels10 cm × 10 cm gels10 cm × 10 cm gels10 cm × 10 cm gels*Recommended for best fit6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com315
Selecting the Best PAGEr Gold Precast GelFresh GelsEvery TimeOrdering Information – PAGEr Gold GelsCat. No. Description SizeSee below PAGEr Gold Tris-Glycine Precast Gels 10 gels per boxGel concentration/separation range4–12% gradient25–250 kDa4–20% gradient5–200 kDa8–16% gradient15–200 kDa10–20% gradient5–150 kDa7.5%50–200 kDa10%25–200 kDa12%20–200 kDa15%10–50 kDaCat. No. Cat. No. Cat. No. Cat. No. Cat. No. Cat. No.Cassette size (cm) 2D well 10 well 12 well 16 well 17 well* 8 + 1 well*9 × 1010 × 109 × 1010 × 109 × 1010 × 109 × 1010 × 109 × 1010 × 109 × 1010 × 109 × 1010 × 109 × 1010 × 10———59557—59564—————59554—59571—59556585205952058511595115851959519585125951258507595075850859508585095950958510595105852259522585055950558521595215850659506585015950158502595025850359503585045950458524595245851759517585235952358518595185851359513585145951458515595155851659516——58545595455856059560——58540—585425954258543595435854459544——58551595515856259562————5854859548——58550595506Electrophoresis and Analysis / Protein316PAGEr Gold Scouting KitPercentage PAGEr Precast Gel■■Gel Concentration and Size Separation Range––Lower concentrations are best for resolving largemolecules and higher concentrations are best forresolving small molecules. Gradient gels are best forproteins that are unknown or occur over a widemolecular weight range.Gel concentration 15 % 12% 10% 7.5% 10–20% 4–20% 8–16% 4–12%Separation pattern225 –225 –225 –225 –225 –225 –50 –225 –50 –225 –50 –50 –50 –Separationsize range10 –15 –50 –25 –Gels were run at 175 volts until the dye front reached the bottom of the gel approximately 60 minutes). 8 µl–10 µl of marker was loaded per lane (0.8 µg–1 µgper band). Gels were stained <strong>with</strong> Coomassie® Brilliant Blue Stain.North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com50 –35 –10–50 kDa 20–200 kDa 25–200 kDa 50–200 kDa 5–150 kDa 5–200 kDa 15–200 kDa 25–250 kDaOrdering Information – PAGEr Gold Scouting KitCat. No. NA Cat. No. EU Product Name Product Description Size58100 58100 PAGEr Gold Scouting Kit 9 cm × 10 cm Select 6 gels f any type59100 59100 PAGEr Gold Scouting Kit, 10 cm × 10 cm 10 cm × 10 cm Select 6 gels f any type5 –5-10 –50 –25 –
PAGEr Minigel ChamberAbsolute Simplicity and Optimal PerformancePAGEr Minigel ChamberThe PAGEr Minigel Chamber is designed to provideoptimized performance from PAGEr Precast Gels and willalso work <strong>with</strong> most other precast minigels. The simple,lock-in-place core design assures a tight, flat fit andeliminates the risk of buffer leaks. No need to remove thecore – simply insert gels, close the clamps, fill <strong>with</strong> bufferand run. Runs one or two gels and accommodates a tankblotting module.■■Benefits––Easy-to-use, lock-in-place core eliminates leaking andminimizes handling––Perfect fit <strong>with</strong> 9 cm × 10 cm and 10 cm × 10 cm PAGErGels––Even electrical force ensures straight lanes––Solid, robust construction––Optimizes performance of PAGEr Gels■■Applications––SDS-PAGE electrophoresis––2D electrophoresis––Tank blottingPAGEr Blot ModuleThe PAGEr Blot Module works directly in the PAGEr MinigelChamber and provides exceptional blotting <strong>with</strong> a fast,simple protocol.■■Benefits––Color-coded cassettes ensure proper orientation of thegel during transfer––Transfer time of 90 minutes or less––Hinged cassette design for easy assemblyThe system can be purchased as a kit, including the PAGErMinigel Chamber and PAGEr Blot Module, or componentsmay be purchased separately.Performance of the PAGEr Minigel ChamberkDa190 –125 –80 –50 –40 –25 –20 –15 –10 –Markers and E. coli lysate run on a 9 cm × 10 cm PAGEr Gel @ 200 V for60 minutes in the PAGEr Minigel Chamber. Samples from left to right: 1and 2 ProSieve Color Protein Marker; 3–8 E. coli lysate; 9 and 10 ProSieveProtein Marker.SpecificationsGel types:Gel sizes:Chamber capacity:Buffer volume:1 2 3 4 5 6 7 8 9 10Most standard precast minigels(casting apparatus not included)9 cm × 10 cm (adapter included) and 10 cm × 10 cmSingle gel (blank included), 2 gels, or blot cassettes≈800 mlwww.lonza.com/sourcebookOrdering Information – PAGEr Minigel ChamberCat. No. NA Cat. No. EU Product Name Product Description Size59905 59905 PAGEr Minigel Chamber 9 cm × 10 cm or 10 cm × 10 cm59906 59906 PAGEr Blot Module each59907 59907 PAGEr Minigel Chamber and Blot Module Kit Includes chamber, 2 blotting cassettes, and sponge 9 cm × 10 cm or 10 cm × 10 cmpads (8/pack).Contact Scientific Support for information about replacement parts.6Electrophoresis and Analysis / ProteinRelated ProductsPageProSieve EX Running and/or Western Blot Transfer Buffer(s) 314PAGEr EX Gels and PAGEr Gold Gels 312AccuGENE Buffers 304Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com317
ProSieve Color Protein MarkersSharp, Accurate Confirmation of Protein Transfer6Electrophoresis and Analysis / ProteinProSieve Color Protein Markers are ideal for monitoringprotein separation prior to staining and provide accurateconfirmation of protein transfer in Western blotting.■■Benefits––Convenient – Just add water and load (ProSieve Coloronly; not required for ProSieve QuadColor)––Sharp – Multi-colored, readily identifiable band patternfor monitoring electrophoresis and confirming proteintransfer––Verify protein transfer following Western blottingProSieve Color Protein Markers are a set of proteins anddyes for use as visible markers in SDS-PAGE gels. Duringelectrophoresis, these markers help monitor the efficiencyof separation. In Western blotting, they confirm transfer hasoccurred from the gel to the membrane. The proteins havebeen labeled <strong>with</strong> fluorescent dyes and contain the buffersalts and detergent found in the typical Laemmli buffersystem.■■ProSieve Color Protein Marker, 10–190 kDa––9 proteins (10, 15, 20, 25, 40, 50, 80, 125, 190 kDa)■■ProSieve QuadColor Protein Marker, 4.6–300 kDa––12 proteins (4.6, 10, 15, 25, 40, 55, 70, 100, 140, 170,250, 300 kDa)NOTE: Not recommended for accurate protein sizing. For sharp, accuratesizing, use ProSieve Protein Markers (page 300).-20°CProSieve Color Protein Markers190 –125 –80 –50 –40 –– 300– 250– 170– 140– 100– 70– 55– 4025 –– 2520 –– 1515 –10 –– 10– 4.6Typical ResultsProSieve Color Protein Marker Performance vs.Leading CompetitorskDa184 –121 –85 –52 –41 –27 –21 –14 –11 –– 300– 250– 170– 140– 100– 70– 55– 40Markers were run on a Lonza 4–20% PAGEr Gold Precast Gel inTris-Glycine SDS Buffer at 200 V for ~60 minutes.Lane 1: Bio-Rad® Precision Plus Dual Color StandardLane 2: Sigma ColorBurst Electrophoresis MarkerLane 3: Lonza ProSieve Color Protein MarkerLane 4: Lonza ProSieve QuadColor Protein MarkerLane 5: Invitrogen BenchMark Pre-Stained LadderLane 6: Invitrogen Novex® Sharp Pre-Stained StandardLane 7: GE Full Range Rainbow® MarkerLane 8: Pierce 3-Color Pre-Stained MarkerLane 9: Lonza ProSieve Color Protein MarkerLane 10: Lonza ProSieve QuadColor Protein MarkerLane 11: Invitrogen SeeBlue® Plus 2 Pre-Stained StandardLane 12: Bio-Rad® Precision Plus Kaleidoscope StandardOrdering Information – ProSieve Color Protein MarkerCat. No. NA Cat. No. EU Product Name Product Description Range Application Size50552 50552 ProSieve Color Protein Marker 10 kDa – 190 kDa 10 100 µl50550 50550 ProSieve Color Protein Marker 10 kDa – 190 kDa 50 500 µl00193837 00193837 ProSieve QuadColor Protein Marker 4.6 kDA – 300 kDA 50 500 µl– 25kDa– 15– 10– 4.6318North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ProSieve Protein MarkersSharp, Accurate Sizing of Proteins 5 kDa–225 kDaProSieve Protein Markers consists of a novel set of proteinsdesigned for accurate sizing of protein samples in SDS-PAGE.Markers contain proteins <strong>with</strong> exact masses and a 50 kDaband of higher intensity for easy identification.■■Benefits––Simple – Wide distribution of exact masses simplifiessample determination––Accurate – Recombinant proteins do not containoligosaccharides that can cause anomalous migration,heterogeneous “fuzzy” bands, and inaccurate sizeestimation––Versatile – Before Western blotting, markers can bevisualized in gel <strong>with</strong> SYPRO® Tangerine Gel Stain(page 304) <strong>with</strong>out inhibition of protein transferPerformance of ProSieve Protein Markers225 –150 –100 –75 –50 –35 –25 –5–225 kDa 10–200 kDa15 –10 –5 –– 200– 150– 120–100– 70– 50– 40– 30– 20– 15– 10-20°COrdering Information – ProSieve Protein MarkerCat. No. NA Cat. No. EU Product Name Range Application Size50547 50547 ProSieve Protein Marker 5 kDa – 225 kDa 100 500 µl00193839 00193839 ProSieve Unstained Protein Marker II 10 kDA – 200 kDa 100 500 µlProSieve ProTrack Dual Color Protein Loading BufferProtect and Track Protein Samples■■Benefits––Protects proteins from degradation during samplepreparation––Two colors for tracking electrophoresis progress (blue)and monitoring Western transfer (pink)––Contains SDS and DTT for complete protein denaturingGelBlot1 2 3 4ProSieve ProTrack Blue Dye monitors protein separation on the gel,while the pink dye confirms transfer of the proteins onto the blot.1 2 3 4Lanes 1 and 3 are proteins protected byProSieve ProTrack Dual Color LoadingBuffer, Lanes 2 and 4 are proteins preparedand run in a standard loading buffer.Ordering Information – ProSieve ProTrack Dual Color Protein Loading BufferCat. No. NA Cat. No. EU Product Name Application Size00193861 00193861 ProSieve ProTrack Dual Color Protein Loading Buffer (4X) 1000 loads 5 ml6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com319
AccuGENE Protein Electrophoresis BuffersOptimum PerformanceAccuGENE Electrophoresis Buffers are formulated tomatch PAGEr Precast Gels. AccuGENE Buffers for proteinelectrophoresis are prepared <strong>with</strong> high quality reagents anduse 18 megOhm water. Products are filtered using a0.2-micron filter.18°C–24°Cwww.lonza.com/sourcebook■■Benefits––Reliable – Manufactured according to strict qualitycontrol standards to ensure lot-to-lot consistency––Efficient – Ready-to-use solutions eliminate preparationtime––Flexible – Customized solutions are available to meetindividual needsOrdering Information – AccuGENE 10X Tris-Glycine BufferCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size50879 BE50879 AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine 18°C–24°C 1 l50881 50881 AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine 18°C–24°C 4 l50882 50882 AccuGENE 10X Tris-Glycine SDS Buffer 0.25 M Tris base, 1.92 M Glycine, 1% SDS 18°C–24°C 4 l6Related ProductsPagePAGEr Minigel Chamber 317PAGEr Precast Gels 296PAGEr Gold Precast Gels 315ProSieve Blue Protein Staining SolutionBelowProSieve ProTrack Dual Color Protein Loading Buffer 319Electrophoresis and Analysis / ProteinProSieve Blue Protein Staining SolutionImproved Coomassie® Dye■■Benefits––Ready-to-use in a fast, 25–40 minute protocol––No destaining required, does not contain methanol oracetic acid––Proteins stain to an endpoint, overstaining is notpossible––Detect 2–5 ng of protein––Broad, linear dynamic range––Can be reused up to three times <strong>with</strong>out a decrease insensitivity500 250 125 63 31 15 8 4 2 ngComparison of detection sensitivity between standard Coomassie® Stain(upper bands) and ProSieve Blue Protein Staining Solution (lowerbands).Ordering Information – ProSieve Blue Protein Staining SolutionCat. No. NA Cat. No. EU Product Name Size00193862 00193862 ProSieve Blue Protein Staining Solution 1 l320North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
SYPRO® Protein Gel StainsFast, Sensitive, Easy-to-use Protein Gel StainsSYPRO® Protein Gel Stains are simple, sensitive alternativesto Coomassie® Brilliant Blue Stain and Silver Stain for adiverse range of applications from 2D gel staining tostaining gels prior to Western blotting.■■Benefits––Exquisitely sensitive – Detection limits rival the bestsilver stains––Fast and easy – Simple procedures require no complexfixation or destain––Quantitative – Broad linear range over 3 orders ofmagnitude––Versatile – Visualize <strong>with</strong> UV transilluminators, DarkReader® transilluminators, and laser scanners––Compatible – With downstream processing such asmass spectrometry and microsequencingSelect the Best Stain for Your ApplicationSensitivity of SYPRO® Stains Compared to Coomassie®Brilliant Blue and Silver StainSerial dilutions of ProSieve Protein Marker 50 kDa band on12% PAGEr Gold Precast Gels, stained and photographed asnoted. Protein levels indicated in nanograms.Coomassie® Brilliant Blue Stain (CCD camera)2.0 3.9 7.8 15.6 31.3 62.5 125 250 500 1000SYPRO® Red Protein Gel Stain diluted in 7.5% acetic acid (Polaroid® Photo UVlight)2.0 3.9 7.8 15.6 31.3 62.5 125 250 500 1000SYPRO® Tangerine Protein Gel Stain diluted in PBS (Polaroid® Photo UV light)2.0 3.9 7.8 15.6 31.3 62.5 125 250 500 1000ApplicationSYPRO®RubySYPRO®TangerineSYPRO®RedHigh performance staining ■ ■Staining prior to Western blotting ■2D Electrophoresis ■Edman microsequencing ■ ■ ■Mass spectrometry ■ ■ ■Quantitation ■ ■ ■Zymography ■ ■ ■Electroelution ■ ■ ■Membrane staining ■Protein expression ■Detection prior to■■ImmunostainingDifficult to stain proteins ■IEF Gels ■Fast, Simple Staining ProcedureStainWashFixation is required for staining 2D gels in SYPRO® Ruby Gel Stain. No washstep is necessary for SYPRO® Red or Tangerine Gel Stains.Related ProductsSilver Stain (Amersham PlusOne Kit)2.0 3.9 7.8 15.6 31.3 62.5 125 250 500 1000SYPRO® Ruby Stain2.0 3.9 7.8 15.6 31.3 62.5 125 250 500 1000PAGEr Precast Gels 315PAGEr EX Gels 312ProSieve Blue Protein Staining Solution 320Page6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com321
SYPRO® Ruby Protein Gel StainThe Best Stain for 2D Gel AnalysisSYPRO® Ruby Protein Gel Stain is a highly sensitive, simpleto use fluorescent protein gel stain that can accuratelyquantitate protein expression levels and is compatible <strong>with</strong>standard fluorescent visualization systems anddownstream identification techniques, such as massspectrometry.■■Benefits––Highly sensitive – Rivals the best silver stain––Quantitative – Broad linear range and consistent gel-to-gel staining––Fast – Simple staining procedure saves time andmoney––High-throughput – Fast, easy staining of multiple gels––Versatile – Detects difficult to stain proteins18°C–26°CSYPRO® Ruby vs. Silver Stain for 2D AnalysisProteins from a cell lysate were run on a 2D gel and stained <strong>with</strong> SYPRO®Ruby Gel Stain (left) or silver stain (right)6Ordering Information – SYPRO® Ruby Protein Gel StainCat. No. NA Cat. No. EU Product Name Product Description Size50564 50564 SYPRO® Ruby Protein Gel Stain 200 ml50562 50562 SYPRO® Ruby Protein Gel Stain Ready-to-use, single reagent format, stains approximately 20 minigels or 2 large 2D gels. 1 l50563 50563 SYPRO® Ruby Protein Gel Stain Ready-to-use, single reagent format, provided in a box <strong>with</strong> a spigot for dispensing. 5 lProduct licensed from Molecular Probes, IncSYPRO® Red Protein Gel StainThe Fastest, Easiest Stain for Detecting ProteinsElectrophoresis and Analysis / ProteinSYPRO® Red Protein Gel Stain is a fast, highly sensitivefluorescent protein gel stain that detects as little as4 ng–8 ng protein per band.■■Benefits––Fast – Complete staining in less than 1 hour––Sensitive – Five times more sensitive than Coomassie®Brilliant Blue Stain––Simple – No fixation or destaining required––Consistent – Low protein-to-protein variabilityStaining is easy – simply soak gels in a solution of 1XSYPRO® Red Stain in 7.5% acetic acid for 40 to 60 minutes.The stain is compatible <strong>with</strong> UV transilluminators, CCDcameras or laser scanners.Photographic filters recommended. See page 324 forproduct deteilsSYPRO® Red Gel StainSDS Polyacrylamide gel stained <strong>with</strong> SYPRO® Red Gel Stain18°C–26°CPage 460Ordering Information – SYPRO® Red Protein Gel StainCat. No. NA Cat. No. EU Product Name Product Description Size50542 50542 SYPRO® Red Protein Gel Stain 10 × 50 μl as a 5,000X concentrate, sufficient for staining approximately 50 minigels. 10 × 50 µl50543 50543 SYPRO® Red Protein Gel Stain 500 μl as a 5,000X concentrate, sufficient for staining approximately 50 minigels. 500 µlProduct licensed from Molecular Probes, Inc322North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
SYPRO® Tangerine Protein Gel StainIdeal for Staining Gels Prior to Western BlottingSYPRO® Tangerine Protein Gel Stain is a versatile, sensitivestain that can be used to visualize proteins prior to Westernblotting.■■Benefits––Visualize proteins prior to transfer – Does not interfere<strong>with</strong> protein activity or transfer––Safe – No acids or organic solvents necessary––Sensitive – Detects as little as 4 ng–8 ng protein perbandThe staining procedure is fast and simple and does notrequire the use of organic solvents; staining can beperformed in saline or PBS solutions. Proteins can be usedin zymography assays or analyzed by mass spectrometry.Performance of SYPRO® Tangerine Gel StainABTwo identical SDS-PAGE gels were run <strong>with</strong> samples of protein molecularweight standards (leftmost lanes) and protein molecular weightstandards mixed <strong>with</strong> decreasing amounts of E. coli ß-glucuronidase andrabbit liver esterase. Gels were stained for total protein <strong>with</strong> SYPRO®Tangerine Protein Gel Stain, and for specific enzymatic activities. Both gelswere first stained <strong>with</strong> SYPRO® Tangerine Protein Gel Stain (one gel shown,Panel A). One gel was stained <strong>with</strong> ELF®-97 ß-d-glucuronidase substrate(E-6587) for the detection of ß-glucuronidase activity (Panel B).18°C–26°COrdering Information – SYPRO® Tangerine Protein Gel StainCat. No. NA Cat. No. EU Product Name Product Description Size50556 50556 SYPRO® Tangerine Protein Gel Stain Supplied as a 5,000X concentrated solution in DMSO, sufficient for staining 50 minigels 500 µlProduct licensed from Molecular Probes, IncSYPRO® Ruby Protein Blot StainFast, Simple, Sensitive Stain for Detecting Proteins on BlotsSYPRO® Ruby Protein Blot Stain offers sensitivity levels thatrival colloidal stains. The stain is 60-times more sensitivethan reversible stains like Ponceau S, and 30-times moresensitive than Amido Black or Coomassie® Brilliant BlueStains.■■Benefits––Highly Sensitive – Detects as little as 2 ng–8 ng proteinper band––Fast – Simple staining procedure takes less than 1 hour––Compatible – With fluorogenic, chemiluminescent andcolorimetric detection techniques18°C–26°CTotal Protein Detection <strong>with</strong> SYPRO® Ruby Protein BlotStainMolecular weight standards containing decreasing amounts of α-tubulinwere run on an SDS-PAGE gel, blotted onto a PVDF membrane and stained<strong>with</strong> SYPRO® Ruby Protein Blot Stain.Ordering Information – SYPRO® Ruby Protein Blot StainCat. No. NA Cat. No. EU Product Name Product Description Size50565 50565 SYPRO® Ruby Protein Blot Stain 200 mlProduct licensed from Molecular Probes, Inc6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com323
SYPRO® Protein Gel Stain Photographic FilterFor Optimal Detection Sensitivity <strong>with</strong> Black and White Film PhotographyThe SYPRO® Protein Gel Stain Photographic Filter is suitablefor Polaroid® Camera Systems. The filter does not work <strong>with</strong>CCD camera systems. Check <strong>with</strong> the manufacturer for theappropriate filter. Recommended for use <strong>with</strong> all SYPRO®Protein Gel Stains.Ordering Information – SYPRO® Protein Gel Stain Photographic FilterCat. No. NA Cat. No. EU Product Name Product Description Size50540 50540 SYPRO® Protein Gel Stain Photographic Filter Wratten® #9 Gelatin Filter 3 inch squareRelated ProductsPagePAGEr Minigel Chamber 317PAGEr EX Gels 312ProSieve Protein Marke 319ProSieve ProTrack Dual Color Protein Loading Buffer 319ProSieve Blue Protein Staining Solution 3206Electrophoresis and Analysis / Protein324North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
IsoGel Agarose and Precast IsoGel Agarose IEF PlatesIsoelectric Focusing for Rapid Separation of Large ProteinsSeparation of proteins in complex mixtures for analyticalresolution can be achieved by isoelectric focusing (IEF), inwhich proteins are separated based on their net charge(isoelectric point or pI) in the presence of a pH gradient.Agarose has distinct advantages over polyacrylamide gelsfor isoelectric focusing. Separation in agarose is more rapid,and agarose gels can be used to separate proteins up to2,000 kDa. We have developed two high quality productsthat are specifically designed and tested for theirperformance <strong>with</strong> IEF.––IsoGel Agarose is a highly purified agarose that is easyto prepare and produces a gel <strong>with</strong> high clarity and aless restrictive matrix than polyacrylamide––IsoGel Agarose IEF Plates are ready-to-use precastgels supported on GelBond® Film, eliminating gelpreparation time and providing easy handlingthroughout the IEF process■■Benefits––Safe – No toxic acrylamide required––Fast – Shorter staining times––Simple – Nontacky and easy to blot■■Applications––Isoelectric focusing––Antibody separation and analysis––Immunofixation directly in the gel––Crossed immunoelectric focusing––Direct tissue or preparative isoelectric focusing––Protein blotting––Immunodetection of proteinswww.lonza.com/sourcebookIsoGel AgaroseHighly Purified Agarose for Isoelectric Focusing■■Benefits––No measurable EEO – Manufacturing process minimizesfixed anions and mobile cations––Versatile – Sufficiently rigid for casting in vertical tubes(e.g., O’Farrell gels), vertically molded or horizontallyopen cast thin gels■■Applications––Isoelectric focusingReferenceO’Farrell, P.H. (1975) High resolution two-dimensional electrophoresisof proteins. J. Biol. Chem. 250: 4007–4021.Analytical SpecificationsMoisture: ≤10%Sulfate:≤0.20%EEO (–m r):Not detectableGel strength (1.5%): ≥500 g/cm 2IEF test:Passes test18°C–26°Cwww.lonza.com/sourcebookOrdering Information – IsoGel AgaroseCat. No. NA Cat. No. EU Product Name Product Description Size50202 50202 IsoGel Agarose For use in isoelectric focusing 25 gLarger package sizes are available upon request. Please inquire for pricing and availability.Related ProductsPageGelBond® PAG Support Film Sheets 307-3086Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com325
Precast IsoGel Agarose IEF PlatesPrecast Gels for the Analysis of Antibodies and Proteins up to 2,000 kDa■■Benefits––Easy handling – Each gel is supported on GelBond® Filmto provide dimensional stability throughout IEFprocessing––Versatile – Convenient 125 mm × 100 mm gel size fitsmost horizontal IEF chambers––Fast – Proteins can be quickly transferred from gel tomembrane, stained in situ, or detected by antibodies<strong>with</strong>in 1 hour■■Applications––Isoelectric focusing––Antibody separation and analysis■■Performance and Quality Tests––Each lot of IsoGel Agarose IEF Plates is functionallytested; Certificate of Analysis available upon requestPerformance of IsoGel Agarose IEF PlateProteins –Lentil lectin –Equine myoglobin –Bovine carbonic –anhydraseOvalbumin –Amyloglucosidase –1 2 3 4 5pI– 8.2– 8.0– 7.8– 7.0– 6.0– 4.8– 3.62°C–8°C for 12 months from the date of manufactureAccessories: 18°C–26°Cwww.lonza.com/sourcebookSeparation of proteins in an IsoGel Agarose IEF Plate, pH 3–10. Lanes1 and 4: pI Marker (in-house). Lanes 2 & 3: Broad Range pI 4.45–9.6marker (Bio-Rad®). Lane 5: Hemoglobin, HB Type AFSC (PE Wallac). 2.5 µlof each sample were loaded on the gel and prefocused at 1 watt for 10minutes and focused at 2000 volts (max), 25 mA (max), 25 W (max) for60 minutes on a GE Multiphor® II Chamber at 10°C. The gel was stained <strong>with</strong>Crowle’s stain.6Electrophoresis and Analysis / ProteinOrdering Information – Precast IsoGel Agarose IEF PlatesCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions Size56015 56015 Precast IsoGel Agarose IEF Plates pH range 3–10 2°C–8°C 6 plates56018 56018 Precast IsoGel Agarose IEF Plates pH range 6–10.5 2°C–8°C 6 plates56024 56024 Precast IsoGel Agarose IEF Plates pH range 7–11 2°C–8°C 6 plates56014 56014 Precast IsoGel Agarose IEF Plate,Contains masks, 100 mm and 125 mm 18°C–26°CSufficient for 6 platesAccessory Packwicks and blotting paper56010 56010 Precast IsoGel Agarose IEF Plate Accessory Contains 125 mm wicks and blotting paper 18°C–26°C 100 eachBulk Pack56007 56007 Precast IsoGel Agarose IEF Blotting Paper 18°C–26°C 250 sheetsRelated ProductsPageGelBond® PAG Support Film Sheets 307-308326North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Agarose for Protein SeparationSafe and Easy Separation of Large Proteins and Protein ComplexesElectrophoresis of proteins in agarose gels has distinctadvantages compared to polyacrylamide for someapplications. Agarose gels can easily and effectivelyseparate high molecular weight proteins and proteincomplexes (>600 kDa).■■Benefits––Safe – No toxic monomer solutions required––Efficient recovery – High recovery yields <strong>with</strong> simpleprocedures––Flexible – Gels can be made <strong>with</strong> standard Laemmlibuffer systems■■Applications––Separation of large proteins and protein complexesNormal Distribution of Von Willebrand FactorMultimers as Separated on 1%, 2% or 3% SeaKem®HGT(P) Agarose Gels278 kDa subunits (n)30 –20 –10 –kDa– 4448– 2780– 1668– 1112– 713– 556– 46918°C–26°Cwww.lonza.com/sourcebookReferences1. Warren, C.M. et al. (2003) Vertical agarose gel electrophoresis andelectroblotting of high molecular weight proteins, Electrophoresis24(11): 1695–1702.2. Smejkal, G.B. et al. (2003) Rapid high-resolution electrophoresis ofmultimeric von Willebrand Factor using a thermopiloted gel apparatus.Electrophoresis 2003, 24: 582–587Protein Separation1.0 2.0 3.0Agarose concentration (%)vWF is the largest protein in the human plasma, the basic repeating unit ofwhich is a 556 kDa homodimer comprised of identical 278 kDa subunitsdisulfide-linked at the C-termini. Each band subfractionates into threebands at higher gel concentrations. Gels prepared <strong>with</strong> 200 mM Tris, 100mM glycine pH 9.0 supplementing <strong>with</strong> 0.1% LiDS. Gels run for 20 min at10°C using 250 V. Proteins were transferred to nitrocellulose membranesand sequentially probed <strong>with</strong> anti-human vWF antisera and alkalinephosphatase secondary antibody and visualized using an enhancedNBT-BCIP substrate. (Used by permission from Gary B. Smejkal, ClevelandState University, Cleveland, Ohio.)Routine Protein Separation Agarose Typical Application Protein Size Range (kDa) Gel ConcentrationMetaPhor Agarose Protein electrophoresis 20–200 4%MetaPhor Agarose Protein electrophoresis 150–300 3%MetaPhor Agarose Protein electrophoresis 300–600 2%SeaKem® Gold Agarose or Protein electrophoresis 600–1,000 1.5%SeaPlaque Agarose Protein electrophoresis 1,000–5,000 1%Specialty Protein SeparationIsoGel Agarose Isoelectric focusing Separation based on isoelectric pointSeaKem® HGT Agarose Counter-immunoelectrophoresis, CIEP, Crossed-IEPSeaKem® HGT(P) Agarose Factor VIII/von Willenbrand’s factor multimer separationSeaKem® ME Agarose Serum protein electrophoresisSeaKem® HEEO Agarose Immunoelectrophoresis of IgG and IgMSeaKem® HE Agarose Serum protein electrophoresis, IEP, Crossed-IEP ,CIEP2 –6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com327
Agarose for Protein SeparationContinuedAnalytical SpecificationsSeaKem® HGT SeaKem® HGT(P) SeaKem® HE SeaKem® HEEO SeaKem® MEGelling temp. (1.5%): 42°C ± 1.5°C 42°C ± 1.5°C 36°C ± 1.5°C 36 ± 1.5°C 36 ± 1.5°CMoisture: ≤10% ≤10% ≤10% ≤10% ≤10%Sulfate: ≤0.30% ≤0.20% ≤0.20% ≤0.25% ≤0.20%EEO (–mr): ≤0.10 ≤0.10 0.23–0.26 ≥0.30 0.16–0.19Gel strength (1%): ≥800 g/cm 2 ≥1,000 g/cm 2 ≥650 g/cm 2 ≥650 g/cm 2 ≥1,000 g/cm 26Electrophoresis and Analysis / ProteinOrdering Information – Agarose for Protein SeparationCat. No. NA Cat. No. EU Product Name Product Description Size50101 50101 SeaPlaque Agarose A low melting alternative for separating proteins ≥600 kDa. 25 g50100 50100 SeaPlaque Agarose A low melting alternative for separating proteins ≥600 kDa. 125 g50014 50014 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 500 g50011 50011 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 25 g50010 50010 SeaKem® ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis 125 g50050 50050 SeaKem® HGT(P) Agarose 125 g50041 50041 SeaKem® HGT Agarose High gelling temperature, high clarity agarose for use in counter-immunoelectrophoresis and 25 gcrossed immunoelectrophoresis50040 50040 SeaKem® HGT Agarose High gelling temperature, high clarity agarose for use in counter-immunoelectrophoresis and 125 gcrossed immunoelectrophoresis50031 50031 SeaKem® HEEO Agarose A very high EEO agarose useful in applications requiring significant cathodal migration, such as 25 gimmunoelectrophoresis of IgG and IgM. May also be blended <strong>with</strong> lower EEO agarose to achieve aspecific EEO value.50030 50030 SeaKem® HEEO Agarose A very high EEO agarose useful in applications requiring significant cathodal migration, such as 125 gimmunoelectrophoresis of IgG and IgM. May also be blended <strong>with</strong> lower EEO agarose to achieve aspecific EEO value.50021 50021 SeaKem® HE Agarose A high EEO agarose that provides enhanced resolution in immunoelectrophoresis, crossed 25 gimmunoelectrophoresis, counter-immunoelectrophoresis, and serum protein electrophoresis.50020 50020 SeaKem® HE Agarose A high EEO agarose that provides enhanced resolution in immunoelectrophoresis, crossed 125 gimmunoelectrophoresis, counter-immunoelectrophoresis, and serum protein electrophoresis.50152 50152 SeaKem® Gold Agarose Effective for separating proteins ≥600 kDa 25 g50150 50150 SeaKem® Gold Agarose Effective for separating proteins ≥600 kDa 125 g50181 50181 MetaPhor Agarose Effective for separating proteins ≥600 kDa 25 g50180 50180 MetaPhor Agarose Effective for separating proteins ≥600 kDa 125 g50202 50202 IsoGel Agarose For use in isoelectric focusing 25 gRelated ProductsProSieve EX Running Buffer 314ProSieve EX Transfer Buffer 314AccuGENE 1 M Tris HCl Buffer 304GelBond® PAG Support Film Sheets 307-308Precast IsoGel Agarose IEF Plates 326Page328North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
ProSieve 50 Acrylamide Gel SolutionModified Acrylamide Formulation for High Performance Electrophoresis of Large Proteins■■Benefits––Gradient separation – From easy-to-cast single concentrationgels––Easy-to-handle – Gels are more durable than standardacrylamide––Sharp resolution – Resolves large proteins (>200 kDa)––Fast – Shorter destaining times and faster proteinmobility times––Low background – Even when used <strong>with</strong> silver stain■■Applications––Protein gel electrophoresis18°C–26°CPage 475www.lonza.com/sourcebookOrdering Information – ProSieve 50 Acrylamide Gel SolutionCat. No. NA Cat. No. EU Product Name Product Description Size50617 50617 ProSieve 50 Acrylamide Gel Solution 50% concentration 125 ml50618 50618 ProSieve 50 Acrylamide Gel Solution 50% concentration 250 ml6Electrophoresis and Analysis / ProteinEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com329
Notes6Electrophoresis and Analysis / Nucleic Acid330North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>7 Services7Cell Services 333Testing Services 341
ServicesCell ServicesIntroduction334Cells on Demand Cell Culture Services 336Cells on Demand Transfection Services 337Clonetics Conditionally Immortalized Cell Lines 338Pluripotent Stem Cell Services 339Testing ServicesQC Testing Solutions Services 342Endotoxin Detection Testing Services 343Mycoplasma Detection Testing Services 346Recertification Services 347Sterilization Services 348Genetically Modified Organism Testing Services 3497332 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell ServicesCell ServicesIntroduction334Cells on Demand Cell Culture Services 336Cells on Demand Transfection Services 337Clonetics Conditionally Immortalized Cell Lines 338Pluripotent Stem Cell Services 3397Services / Cell ServicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com333
IntroductionDiseaseDiscoveryTargetIdentificationand ValidationHitFindingLeadOptimizationPreclinicalADMETClinicalTrialsCommercialProductionRelevant AccelerationWe have a reputation for expertise in many areas of cellbiology, from the earliest research throughbiopharmaceutical production. Our presence in manypharmaceutical product and service markets helps us tounderstand the limitations of existing technologies andguides our efforts to find and develop better solutions forour customers.Lonza <strong>Research</strong> Solutions is the market leader in primarycell biology. Over the past decade, the use of primary cellshas been expanding. As primary cells are more difficult tohandle than cell lines, we deliver complete protocols toaddress this. The importance of consistent tools in drugdiscovery is indisputable, and we strive to standardize ourhighly relevant products.Our SolutionsWe focus on the following areas of product and servicedevelopment.––Primary cell supply, including diseased cells, stem cellderivedcells, iPSC generation and banking,immortalized cells, cells containing biosensors,transfection of difficult cells and cell expansion services––Improved prediction from cell models, includingcontextual cell-based assays for toxicity andmechanism of action assessment––Making it easier for you, including reagent production,Cells on Demand and Pluripotent Stem Cell Servicesand putting the assays you already use into biologicallyrelevant primary cells7Our dedicated Drug Discovery Team gives you access to ourexpertise in many areas of cell biology. We trust that wehave identified the products and services of value to drugdiscovery customers and we welcome the opportunity todiscuss and develop them further <strong>with</strong> you.Services / Cell Services334 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Clonetics Cell ModelsRenal Epithelial Cell ModelClonetics Renal Epithelial Cell Model recreates human renaltubular function in an in vitro environment. The modelconsists of primary epithelial cells isolated from humanrenal cortices and are seeded on extracellular matrixcoatedpolyester membrane inserts, which allows for apicaland basal exposure to culture medium.■■Benefits––Non-transformed, non-immortalized normal humanrenal epithelial cells––Expression of apical brush border enzyme gammaglutamyltranspeptidase (γ-GTP) for 14 daysRenal epithelial cells stained for ZO-1 cellular junction protein■■Applications––Active and passive transport functions––Nephrotoxicity evaluation and renal tubular biologyg-GTP is Expressed Primarily on Apical SurfaceγGTP Molar ratio (apical:basal)1,00010010Relative Fluorescence UnitsBasolateralate al Up ak oUptakeOrgani CaofnsOrganic Cations8641Lot 1 Lot 2 Lot 3Ratios of apical-to-basal expression of γ-GTP were averaged for 3 lots ofrenal cortical epithelial cells in 2 separate experiments (triplicate wells foreach lot).20Lot 1 Lot 2 Lot 3Apical UptakeBasal UptakeUptake of rhodamine 123 was assessed from both apical and basal culturemedium. Fluorescence of extracted rhodamine 123 was normalized toapical concentration in each experiment. Results are from 3 lots of renalcortical epithelial cells (triplicate wells for each lot) in 2 separateexperiments.Ordering Information – Cell ModelsCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeCMS-2000 CMS-2000 Renal Epithelial Cell Model Plated cells – Polyester Transwell® Membranes 2°C - 8°C 12 transwell membranesOrdering Information – Epithelial Cell MediaCat. No. NA Cat. No. EU Product Name Product Description Storage Conditions SizeCC-3190 CC-3190 REGM Renal Epithelial Cell Growth Medium BulletKit Includes basal medium and SingleQuots Kit -10°C to -20°C KitCC-3191 CC-3191 REBM Renal Epithelial Cell Basal Medium 2°C - 8°C 500 mlCC-4127 CC-4127 REGM Renal Epithelial Cell Growth Medium SingleQuotsSupplements and Growth Factors-10°C to -20°C Kit7Services / Cell ServicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com335
Cells on Demand Cell Culture ServicesConsult the cell culture experts and harness the power ofour extensive experience in delivering bulk cell productionand cell isolation services. Just select the cell types andformat and we will do the rest. Delays in obtaining vital cellcultures can be very costly. For this reason, we invite you todiscover our superior cell culture capabilities, deliveredon-time and to your specifications.––Cell line bulk production––Primary cell expansion––Primary cell isolations from many species––Cells delivered in a variety of formats■■Benefits––Allows researchers to focus on their experiments––Simplifies primary cell culture––Move to a more biologically relevant cell model––Avoid the aggravation of failed isolations and low yields––Utilize our tissue acquisition networks www.lonza.com/cellsondemand7Services / Cell Services336 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cells on Demand Transfection ServicesWe provide unparalleled transfection services for primarycells and difficult-to-transfect cell lines. The results andexpertise are proven: Nucleofection is used in expressionstudies, RNAi knockdown and shRNA experiments, transientprotein expression, target screening, target validation, andstable clone generation. More than 4,000 scientists are nowusing Nucleofection to help achieve their researchobjectives.––Stable Clone Generation – Pooled or single clones––Nucleofection Protocol Optimization––Small scale protein production up to 1 l culture––Transiently transfected primary cells or cell lines––Ready to use cells cryopreserved in plates■■Benefits––Stable clones generated and validated by experiencedscientists––Achieve high transfection efficiency and viability––Obtain Nucleofection Protocols guaranteed to work<strong>with</strong> your cell types––Save time and money by utilizing our transfectionexpertise––Flexibility in planning experiments <strong>with</strong> ready-to-assaycells cryopreserved in platesD ubl Stabl Jurka 6.1Inducible Reporter in HUVECLuminescence per wellMean Fluorescence Inten1,2001,0008006004002008006004000 0.5 1 5 10 100Forskolin concentration (µM)HUVEC (Lonza CC-2517) were transiently transfected by Nucleofection<strong>with</strong> a cAMP inducible luciferase reporter construct (pGL4.29, Promega)and subsequently treated <strong>with</strong> different Forskolin concentrations.Double Stable Jurkat E6.1800600400Rf sFrozen Cells on PlatesLuminescence per well5,0004,0003,0002,0001,0000 0.5 2.5 5 7.5 10 15Forskolin concentration (µg)Cryopreserved cellsNon-frozen controlDose-response to Forskolin of CHO-CRE-luciferase cells, (pink line)cryopreserved assay ready on 96-well plates in a proprietary Lonzacryoprotective agent, (blue line) non-frozen control cells.2000Clone 24 Clone 28 Clone 34 Clone 39 Clone 51GFPGFP and luciferase expression level. Single-cell derived Jurkat E6.1 cloneswere generated by cotransfection of pmaxFP-green N and a luciferaseplasmid using Nucleofection and limiting dilution.www.lonza.com/TRSLuciferase20007Services / Cell ServicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com337
Clonetics Conditionally Immortalized Cell LinesCell lines are commonly used screening models becauseproduction is easily scalable and these models minimizevariability by providing uniform biological response overscreening campaigns lasting many months. Onedisadvantage is that cell lines often lack the relevantphenotype of a cell in vivo. To address this shortcoming, wedeveloped Clonetics Conditionally Immortalized Cells.These unique cells are generated by combining the telomererejuvenating effects of telomerase <strong>with</strong> a specially modifiedtemperature controlled T-antigen.■■Benefits––Biological relevance of primary cells––Ease-of-use of a cell line––Production is easily scalable––Minimal lot-to-lot variation––Unlimited supply of differentiated cells of samegenotype■■Applications––High-throughput screeningConditional Immortalization37°C – Normal phenotypeCells isolated fromhuman tissuesTransfect <strong>with</strong>Large T-antigenand telomeraseCells are createdexpressing temperaturesensitive Large T-antigenand telomerase33°C – Large T-antigen activeCell populationexpanded due toimmortalizationImmortalized phenotype(gene expression altered)ImmortalizationSwitched ON37°C – Normal phenotypeCells revert to anormal phenotypeImmortalizationSwitched OFF7Services / Cell ServicesOrdering Information – CellsCat. No. Description Proliferation Media MediaXM13A1 Conditionally Immortalized Skeletal Muscle Myoblasts SkGM-2 CC-3245XM15B1 Conditionally Immortalized Skeletal Muscle Myoblasts SkGM-2 CC-3245XA15A1 Conditionally Immortalized Human Preadipocytes SkGM-2* CC-3245XF05C1 Immortalized Human Dermal Fibroblasts DMEM/M199 + GF** –XS12C1 Immortalized Human Coronary Artery Smooth Muscle Cells SmGM-2 CC-3182XSEL6C1 Immortalized Human Lymphatic Microvascular Endothelial Cells EGM-2 MV CC-3202XSEB113C1 Immortalized Human Blood Microvascular Endothelial Cells EGM-2 MV CC-3202XSKA1B1 Conditionally Immortalized Human Adult Dermal Keratinocytes (cloned) KGM-2 CC-3107XSKA9B1 Conditionally Immortalized Human Adult Dermal Keratinocytes (pooled) KGM-2 CC-310700194607 Conditionally Immortalized Human Brain Microvascular Endothelial Cells EGM-2 MV CC-3202See page 381–389*Differentiation media PT-8002**FBS, L-Gluthamine, GA-1000338 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pluripotent Stem Cell ServicesLonza established a new strategic vision to become the leading supplier to the regenerative medicine industry. To realize thisvision, Lonza created the Pluripotent Stem Cell Innovation Center. Pluripotent stem cells (PSCs) have the ability to generate anyof the 220 + cell types in the human body. And because of this unique attribute, these cells have great potential in basic research,drug discovery and cell replacement therapies.iPSCsTissue AcquistionReprogrammingGrowthExpansionCharacterizationDifferentiationGrowthExpansionCharacterizationDifferentiationPSC Services Lonza has built up expertise, capacity, and capabilities in pluripotent stem cell research and their application to cGMP manufacturing. <strong>Research</strong>erscan now access this expertise through our PSC service offering from iPSC generation to process development and differentiation.Our services span the full value chain of pluripotent stemcells from tissue acquisition to differentiation:■■Tissue Acquisition – We have a dedicated team thatprocures both research and cGMP grade tissueaccording to the highest ethical standards and incompliance <strong>with</strong> government regulations.■■Reprogramming – We offer cGMP and non-cGMP iPSCgeneration under feeder- and feeder-free conditionsusing a zero-footprint technology■■Growth / Expansion / Banking – We have establishedprotocols using all of the common medium, matrix, andpassaging methods. We also have the infrastructureand resources to support small- and large-scale cultureand banking of PSCs■■Characterization – We offer all the standard methods ofcharacterizing PSCs including thawing efficiency,myoplasma and sterility testing, karyotype analysis,short tandem repeat genotyping, pluripotency markerexpression (flow cytometry and immunofluorescence),and pluripotency assays (embryoid body and teratomaformation)■■Differentiation – We have established protocols for theproduction of PSC-derive d motor neurons, dopaminergicneurons, and neural stem cells. We also havedevelopment programs underway to add to ourdifferentiation portfolio of therapeutically relevant celltypes■■Process development – Over the years we have built upexpertise in the differentiation of high purity, functionalcell types. Our team is well versed in technology transferand optimization of manufacturing protocolsAlkalinePhosphateAlkalinePhosphateOCT4 SSEA4NANOG DAPILonza iPSCs express hESC-associated markers POU5F1/OCT4 (green) andSSEA4 (red) counterstained <strong>with</strong> DAPI (blue).7Services / Cell ServicesEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com339
Notes7Services / Cell Services340 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Testing ServicesTesting ServicesQC Testing Solutions Services 342Endotoxin Detection Testing Services 343Mycoplasma Detection Testing Services 346Recertification Services 347Sterilization Services 348Genetically Modified Organism Testing Services 3497Services / TestingEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com341
QC Testing Solutions ServicesAnother means by which Lonza continues to strive to meetthe needs of our QC microbiology customers is by providingcomplementary contract testing services for many of ourQC microbiology related products.QC Testing Solutions Services offered:––Endotoxin Testing Services––Mycoplasma Testing Services––Recertification Services––Sterilization Services––Genetically Modified Organism (GMO) Testing ServicesQC Testing Solutions Services are presently operated out ofthree global Lonza facilities, to provide our customers <strong>with</strong>worldwide access to all of our current service capabilities*:––Lonza Walkersville, Maryland, USA––Lonza Verviers, BELGIUM––Lonza Saint Beauzire, FRANCE*Not all testing services are offered in every location.Please contact your local sales representative or CustomerService for more information about where and how tosubmit samples to one of our service locations above.7Register for our QC Testing Solutions e-newsletter for moreinformation about new service capabilities:www.lonza.com/enewsServices / Testing342 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Endotoxin Detection Testing ServicesLonza’s Endotoxin Detection Testing Services offer you thebest in routine and customized endotoxin testing. Ourfacilities are GMP compliant and provide expertise in gelclot, kinetic chromogenic, kinetic turbidimetric LAL, as wellas rFC endpoint fluorescent assays.USP, EP and FDA CompliantAll pharmaceutical grade endotoxin testing is performed inaccordance <strong>with</strong> current regulatory documents such as theU.S. and European Pharmacopeia (USP and EP) BacterialEndotoxins Test (BET), the 2012 U.S. Food and DrugAdministration (USFDA) Guidance for Industry and the USPmonograph for medical device testing. These documentsoriginate from or are acceptable to the major Pharmacopeiaand their regulatory authorities.Whether you are from a university, research laboratory or aQuality Control laboratory in a major pharmaceuticalcompany, Lonza has the capabilities you need for reliable,accurate and confidential results.■■Benefits––Market leader in endotoxin detection testing systems––Technical expertise and full service reporting––USP and EP compliantSTAT Testing Available*For customers who may need faster turn-around times,Lonza offers an expedited testing service for preliminaryscreening, endotoxin determination, and the EndotoxinChallenge Vial test. Our STAT service delivers preliminary(unaudited) results in 4 days or less. STAT requests requireprior approval from Lonza’s Endotoxin Testing Services.*STAT testing is only available in the US.www.lonza.com/endotestservIf your product does notrequire a validation step in ourlaboratoryOffered in U.S. and EuropeEndotoxinTesting ServiceIf your product does notrequire a validation step in ourlaboratory80-501Product validationInhibition/enhancementRelease of product indevelopment phaseValidation on1 batch80-503Endotoxin determination of asingle sample80-500Product preliminaryscreeningMay be required prior totesting under 80-501and 80-50280-502Product validationInhibition/enhancementRelease of GMP product80-504Endotoxin determination ofthree samples of a single batchValidation on3 batches7Services / Testing80-509Endotoxin determination ofadditional samples of sameshipment sent under 80-50380-510Endotoxin determination of threesamples of additional batches sentunder 80-504Figure 1. Endotoxin Testing Service.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com343
Endotoxin Detection Testing ServicesContinuedPre-paid, Mail-in Anytime Testing ServiceAvailable in Europe, the Endotoxin Testing Service offers alow cost mail-in anytime service for users not requiringtesting according to pharmaceutical or medical deviceregulations.This type of endotoxin testing is suitable for a wide range ofnon-standard tests such as:––Samples from cell culture materials––Samples of DNA plasmids––Water samples from autoclaves or dialysis machines––Dialysis water or fluids––<strong>Research</strong> samplesSamples received using this service will be tested at therequired dilutions indicated in the Pre-paid EndotoxinTesting Service diagram.■■Benefits––Low cost, convenient and quick endotoxin testing viaKinetic-QCL LAL Assay––Quick, easy ordering process via web, phone, or fax––Pre-addressed packaging for smooth delivery to Lonza’sEndotoxin Testing ServiceTo utilize the Pre-paid Service, simply order one or more ofthe Rapid Endotest packs, place your sample in the samplecontainers provided, and mail the pre-addressed envelopeback to Lonza.When ordering the catalog numbers for the Pre-paid Service,91-101 for example, the pre-paid endotest packs areincluded to ship the samples to the Endotoxin TestingServices. When you have a requirement for an endotoxintest, simply mail in your samples. Samples will be testedand results sent to the customer <strong>with</strong>in 5 days of receipt.www.lonza.com/endotestservPre-paid1 Sample 5 Samples791-10xTwo specific catalog numbers areavailable depending on the quantity95-10xof samples to be tested.Services / Testing91-101Typically WaterSampleTested 1/1 bydefault91-102Typically DialysisSampleTested 1/5 and1/10 by default91-103Typically<strong>Research</strong> SampleTested 1/10,1/100 and1/1000 by defaultDilutionSchemeThree optionsare availabledepending onthe quantity ofdilutions to betested95-101Typically WaterSampleTested 1/1by default95-102Typically DialysisSampleTested 1/5 and1/10 by default95-103Typically <strong>Research</strong>SampleTested 1/10,1/100 and 1/1000by defaultFigure 2. Pre-paid Testing Service, available in Europe.91-101 and 95-101 are available in US as well.80-508Special TreatmentThe Pre-paid Services do not include special handlingon your product. On request, a specific treatment isapplied or special reagents are used (pH adjustment,heat inactivation, use of Tris-Buffer, MgCI 2,PYROSPERSE or glucan blocker.)344 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Endotoxin Detection Testing ServicesContinuedOrdering Information – Endotoxin Detection Testing ServicesCat. No. NA Cat. No. EU Product Name Product Description80-500 BE80-500 Product preliminary screening80-501 BE80-501 Product validation inhibtion/enhancement Release of product in development phase. Validation onone batch.80-502 BE80-502 Product validation inhibition/enhancement Release of GMP product. Validation on three batches.80-503 BE80-503 Endotoxin determination of single sample80-509 BE80-509 Endotoxin determination of additional samples of same shipment sentunder 80-50380-504 BE80-504 Endotoxin determination of three samples of a single batch80-510 BE80-510 Endotoxin determination of three samples of additional batches sentunder 80-50480-508 BE80-508 Unusual sample treatment or handling charge per hour80-514 BE80-514 Exclusion of β-D-glucan activity testing80-535 BE80-535 Depyrogenation Endotoxin Challenge Vial Test80-544 80-544 Sample container screening80-500S n/a STAT: Product prelimary screening of one batch80-503S n/a STAT: Endotoxin determination of single sample80-509S n/a STAT: Additional samples of same shipment sent under 80-50380-504S n/a STAT: Endotoxin determination of three samples of a single batch80-510S n/a STAT: Endotoxin determination of three samples of additional batchessent under 80-50480-514S n/a STAT: Exclusion of β-D-glucan activity testing80-535S n/a STAT: Depyrogenation Endotoxin Challenge Vial TestSTAT Testing is not available in Europe.Ordering Information – Pre-paid Endotoxin Detection Testing ServicesCat. No. NA Cat. No. EU Product Name Product Description91-101 BE91-101 Rapid-Endotest Single Water SampleBE91-102 Rapid-Endotest Single Dialysis Fluid SampleBE91-103 Rapid-Endotest Single <strong>Research</strong> Sample95-101 BE95-101 Rapid-Endotest Five Water SamplesBE95-102 Rapid-Endotest Five Dialysis Fluid SamplesBE95-103 Rapid-Endotest Five <strong>Research</strong> Samples80-515-X1 15 Endotoxin Challenge Vials Endotoxin testing of baked vials <strong>with</strong> positive product controls. Price includes ECVs.80-515-X2 20 Endotoxin Challenge Vials Endotoxin testing of baked vials <strong>with</strong> positive product controls. Price includes ECVs.80-515-X3 25 Endotoxin Challenge Vials Endotoxin testing of baked vials <strong>with</strong> positive product controls. Price includes ECVs.Please contact your local Customer Service to obtaingeneral information and the appropriate sample submissionforms.7Services / TestingU.S. Endotoxin Testing ServicesE-mail: endotoxin.testing@lonza.comEuropean Endotoxin Testing ServicesE-mail: scientific.support.eu@lonza.comEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com345
Mycoplasma Detection Testing ServicesLonza QC Testing offers rapid mycoplasma testing servicesusing a regulatory accepted PCR-based mycoplasmadetection method. As <strong>with</strong> our Endotoxin Testing Service,we use the same test methods in our own laboratories aswe perform on your samples.7Our mycoplasma testing services include:Test Article Screening – a feasibility study to assesswhether the sample (test article) causes any potential PCRinhibition which could compromise the validity of the testresult. Matrix interference, or PCR inhibition, will beassessed <strong>with</strong> live mycoplasma spiked into the sample. Ascreening test can also be used to determine whether theproduct is suitable for the rapid mycoplasma test, orwhether an alternative testing approach must be taken.It is recommended that all new products undergo ascreening test for a comprehensive overview of the samplebehavior <strong>with</strong> the PCR test.Rapid Assay Qualification (validation) – a validation studyconducted by Lonza, on your behalf and according to yourspecifications, to approve a product for final product releaseusing an alternative rapid mycoplasma testing method.Standard Rapid Mycoplasma Test – processes a research orin-process sample according to the customary procedure ofthe rapid PCR test.All samples submitted for final release must have undergonea complete validation of the rapid mycoplasma PCR test.Although sample requirements may vary according toservice rendered, customers are asked to submit ~30 mlper lot of each product to be analyzed. Actual requiredvolume is dependent on test performed.How to submit samplesPlease contact your local Lonza sales representative orLonza Customer Service department for more informationabout pricing, special discounts, and coordinating shipmentof samples to one of our testing laboratories.Fill out and follow the instructions on the MycoplasmaTesting Services Sample Submission Form supplied by therepresentative and return to Lonza to ensure properdelivery and sample tracking.www.lonza.com/mycotestservServices / TestingOrdering Information – Mycoplasma Detection Testing ServicesCat. No. NA Cat. No. EU Product Name Final Results00199829 00199829 Test Article Screening 2 weeks00199830 00199830 Rapid Mycoplasma Assay Qualification 6 weeks00199831 00199831 Standard Rapid Mycoplasma Test 5 days346 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Recertification ServicesRecertification Service for Stepped Neutral Density and Absorbance Test PlatesThe SND and Absorbance (Universal) Test Plates provide theELx808 user <strong>with</strong> the ability to manage risk and helpassure the reader is functioning properly between therecommended six-month preventive maintenance visits.The ELx808 readers are robust instruments designed toprovide years of reliable performance for endotoxindetection and other absorbance-based assays. In followingcurrent Good Manufacturing Practices (cGMP), the reader’sperformance should be verified and documented at regularintervals.In order to properly assess the reader performance, theplates used in the testing must be shown to be acceptable.The plate manufacturers recommend the plates berecertified at least once per year.■■Benefits––Manages risk and helps ensure optimal performance ofthe ELx808 Readers––cGMP compliant processes, including testing of multipleparameters––NIST-traceable testing is conducted in an ISO 9001certified facility■■Parameters tested––Absorbance accuracy (linearity and slope)––Precision (reproducibility of results)––Wavelength accuracy––Optical alignment––Detailed physical inspection■■Reports include––Physical inspection observations––Coefficient of variation (%CV) for each plate standardfilter*––Slope, R value and Y intercept linearity data*––Reader and plate uncertainty values––Approval by a Quality Assurance representative––Recertification due date (default is 12 months)*Tested but not printed on the Absorbance Test Plate data forms.Please refer to the table below for additional details andordering information. For testing parameters, reports andpricing, please contact your local sales representative.www.lonza.com/recert7Ordering Information – Recertification ServicesCat. No. NA Cat. No. EU Product Name Product Description85-011 85-011 Stepped Neutral Density Plate Recertification Service Two filter wavelengths 405 nm & 340 nm85-012 85-012 Stepped Neutral Density Plate Recertification Service Each additional filter wavelength85-013 85-013 Absorbance Test Plate Recertification ServiceRecertification Service offered for Lonza plate part numbers 25-272 and 25-342 and BioTek Instruments,Inc. plate part numbers 9000547 (Universal Test Plate) and 7260522 (Absorbance Test Plate).Services / TestingEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com347
Sterilization ServicesLonza Walkersville offers Steam and Dry-Heat SterilizationServices for components used in the medical device andpharmaceutical industries. A Complementary BusinessSolution from Lonza’s QC Testing Solutions, sterilizationprocesses are performed following current Quality SystemsRegulations in an ISO 9001:2008 certified facility.Lonza’s large capacity autoclaves allow the flexibility ofhandling almost any size load or packaging configurationused in the medical device industry. Competitive pricingscenarios can be arranged for customized processes.■■BenefitsContract sterilization can be performed on a variety ofpackaging configurations:––Dry goods––Flexible packaged items and liquids (including those insealed glass or plastic containers involving lowtemperature closed terminal sterilization)Verification of sterilization is achieved utilizing the referencebacterial spore, G. stearolthermophilus as a biologicalindicator. All sterilization runs are reviewed by Lonza’sQuality Assurance (QA) department that will issue aCertificate of Sterilization and sign-off on each customerrun.Lonza also offers a depyrogenation service to obtainverified three log reductions in endotoxin levels. Glass vialsfrom 1 ml to 100 ml in size can be certified as ‘depyrogenated’in our large capacity ovens. The process utilizes dry-heat at250ºC for a minimum of 2.5 hours. All depyrogenationservices are performed <strong>with</strong>in an ISO 8/Class 100,000clean room.Please contact Customer Service for additional informationregarding Sterilization Services.7Services / Testing348 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Genetically Modified Organism (GMO) Testing ServicesHelping Ensure the Quality and Integrity of Products along the Entire Food ChainEuropean regulations require the testing of foodend-products and traceability of all ingredients used duringthe production process for the presence of GeneticallyModified Organisms (GMOs) in raw materials. Food suppliersmust meet consumers‘ expectations for safety andwholesomeness, while complying <strong>with</strong> GMO labelingregulations. The European Union mandates that labels mustindicate when GMO content in a food product is above 0.9%.GMO Testing Services, a Complimentary Business Solutionfrom Lonza’s QC Testing Solutions, offers screening,identification, and quantification of genetically modifiedevents in a broad variety of products, such as seeds, food,feed, and final products.■■BenefitsPreserve the identity of your product from seed to finalproduct by:––Screening for the presence/absence of GMO events toensure that no adulteration has occurred––Protecting product integrity and brand value––Quantification of GMOs for accurate labeling of your finalproductTest Name Test Type TurnaroundTimeGMO Alert SilverGMO Alert GoldGMO UltraPlatinumGMO Ultra MaizeGMO Ultra SoyGMO UltraSecondary EventGMO AlertCustomPreliminary Screening ofP35S–TNOSQuantitative ScreeningRush Screening of P35S-TNOSQuantitative ScreeningIdentification and Quantification of allCorn and Roundup Ready® Soy EventsIdentification and Quantification of AllCorn EventsIdentification and Quantification ofRoundup® Ready Soy EventsAdditional Identification andQuantification of all Corn and RoundupReady® Soy Events after PositiveScreening of 700-01 or 700-02Custom Analysis for Other Analysis(tobacco, rapeseed, wheat, etc.) UponRequest48 hrs24 hrs48 hrs48 hrs48 hrs24 hrs48 hrs■■Our GMO Testing Services provides:––Real Time Polymerase Chain Reaction (PCR) basedassays for the detection and quantification of GMOevents in a wide variety of products––Service availability throughout Europe and otherlocations around the world––Strict adherence to regulatory guidelines on reliability,reproducibility, and accuracy––A documented internal proficiency testing program tohelp ensure the highest quality standards and testingintegrityPlease contact your local Customer Service for moreinformation about our GMO Testing Service capabilities.www.lonza.com/gmo7Services / TestingEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com349
Notes7Services / Testing350 North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>8 QC Testing Solutions8Endotoxin Detection 353Instrumentation and Software 373Accessory Products 379
QC Testing SolutionsEndotoxin DetectionIntroduction354Overview of LAL Testing Procedures 355Overview of Endotoxin Detection Methods 356Kinetic Chromogenic LAL Assay Overview 358Kinetic-QCL Kinetic Chromogenic LAL Assay 359Control Standard Endotoxin for Kinetic-QCLBulk Kinetic Chromogenic LAL 359QCL-1000 Endpoint Chromogenic LAL Assay 360Kinetic Turbidimetric LAL Assay Overview 361PYROGENT-5000 Kinetic Turbidimetric LAL Assay 362Reconstitution Buffer for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 362Control Standard Endotoxin for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 363Alternative Method – PyroGene RecombinantFactor C Assay 364PYROGENT Gel Clot LAL Assay Overview 367PYROGENT Gel Clot LAL Assay 368PYROGENT Plus Gel Clot LAL Assay 369PYROGENT Ultra Gel Clot LAL Assay 370PYROGENT Bulk Gel Clot LAL Assay 371Control Standard Endotoxin for PYROGENT Gel Clot LAL 372Instrumentation and SoftwareELx808 Absorbance Plate Reader 374FLx800 Fluorescence Plate Reader 375PyroTec Liquid Handling System 376WinKQCL Endotoxin Detection and Analysis Software 3778Accessory ProductsIntroduction380Test Tubes 380Sample Containers 381Plates381Pipette Tips 382Reservoirs382Dry Heat Block, Inserts and Vortex Mixer 383LAL Reagent Water 383β-G-Blocker384PYROSPERSE384MgCl 2 385Tris Buffer 385Endotoxin and Endotoxin Challenge Vials 386352North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Endotoxin DetectionFast and Easy Endotoxin Detection for Your <strong>Research</strong>Endotoxin DetectionIntroduction354Overview of LAL Testing Procedures 355Overview of Endotoxin Detection Methods 356Kinetic Chromogenic LAL Assay Overview 358Kinetic-QCL Kinetic Chromogenic LAL Assay 359Control Standard Endotoxin for Kinetic-QCLBulk Kinetic Chromogenic LAL 359QCL-1000 Endpoint Chromogenic LAL Assay 360Kinetic Turbidimetric LAL Assay Overview 361PYROGENT-5000 Kinetic Turbidimetric LAL Assay 362Reconstitution Buffer for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 362Control Standard Endotoxin for PYROGENT-5000Bulk Kinetic Turbidimetric LAL 363Alternative Method – PyroGene RecombinantFactor C Assay 364PYROGENT Gel Clot LAL Assay Overview 367PYROGENT Gel Clot LAL Assay 368PYROGENT Plus Gel Clot LAL Assay 369PYROGENT Ultra Gel Clot LAL Assay 370PYROGENT Bulk Gel Clot LAL Assay 371Control Standard Endotoxin for PYROGENT Gel Clot LAL 372QC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com353
IntroductionQC Testing Solutions / Endotoxin Detection8Endotoxin Detection: A Brief HistoryEver since the pharmaceutical industry beganmanufacturing injectables, pyrogen detection tests havebeen an absolute necessity. Pyrogens are substances thatcan cause fever, shock and even death if high levels areintroduced into the body. Endotoxins are natural compoundsfound in the outer cell membrane of Gram-negative bacteriaand are released upon cell lysis. Endotoxins are a type ofpyrogen. Today, endotoxin detection tests are performed onraw and in-process materials and for the final release ofproducts in the pharmaceutical and medical deviceindustries.For most of the 20th century, the rabbit pyrogen test wasthe standard method of testing for pyrogenicity. This test,which takes approximately four hours, is accomplished byinjecting the drug being analyzed into a rabbit’s ear. If theanimal develops a fever, it confirms the presence ofpyrogens.The LAL test was commercially introduced in the 1970s. LALis derived from the blood cells, or amebocytes, of thehorseshoe crab, Limulus polyphemus. LAL was developedinto a test for endotoxin after Frederick Bang and Jack Levinobserved that the amebocytes of the horseshoe crabcontain a clotting agent that gels in the presence of Gramnegativebacteria. They recognized that this clotting agentcould be used as a definitive way to test pharmaceuticaldrugs for the presence of Gram-negative bacteria.In a notice published in the Federal Register on November 4,1977, the FDA described conditions for the use of LAL as anend-product test for endotoxin in human biological productsand medical devices. The FDA noted it is widely recognizedthat the LAL test is faster, more economical and requires asmaller volume of product than does the rabbit pyrogentest. In addition, the procedure is less labor intensive thanthe rabbit test, making it possible to perform many tests ina single day.To obtain the lysate required for the LAL test, horseshoecrabs are taken from the ocean floor and a small amount oftheir blood is drawn. The animals are then returnedunharmed to the sea. Next, the crab’s blood cells,amebocytes, are separated and lysed to obtain the cellularproteins.As LAL became the preferred endotoxin detection test,different methods were developed, each <strong>with</strong> its own uniquebenefits. For example, Gel Clot LAL (PYROGENT) provides asimple positive/ negative result and is mentioned in mostpharmacopeial monographs as the official referee test.Kinetic turbidimetric LAL (PYROGENT-5000) gives aquantitative result, and offers an economical choice forwater and large volume parenterals. Endpoint chromogenicLAL (QCL-1000) offers a quantitative result and exhibitsless product interference than LAL methods utilizing theclotting protein. Kinetic chromogenic LAL (Kinetic-QCL)provides the benefit of less product interference forproteins, vaccines and other biologicals as well as greatersensitivity, detecting as low as 0.005 EU/ml.Currently the FDA, the United States Pharmacopeia (USP),the European Pharmacopeia (EP) and the JapanesePharmacopeia (JP) accept all of the above LAL methods, asdo most individual country pharmacopeias.More recently, Lonza scientists have developed a reliableand sustainable endotoxin detection test method that is notderived from horseshoe crab blood. The PyroGene Assay isbased on the recombinantly expressed Factor C which is thefirst component in the horseshoe crab clotting cascadeactivated by endotoxin. It is specific for endotoxin andoffers a reliable alternative for endotoxin release testing.The PyroGene Assay promises to reduce the dependenceon animal-based endotoxin assays. In 2009, the FDAapproved 510(K) applications that included the PyroGeneAssay as the final release test. The latest FDA guidancedocument for Industry on “Pyrogene and EndotoxinsTesting: Questions and Answers” from 2012 accepts the useof PyroGene as an alternative method. Please refer to page364 for further information.www.lonza.com/lal354North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Overview of LAL Testing ProceduresThere are four basic types of assays, each of which isdesigned to perform a different aspect of LAL testing.Our WinKQCL 5 Software supports all of these assay typesand is the ideal tool to accompany your quantitativeendotoxin assays. It offers a fully integrated solution forreporting and analyzing your endotoxin assay results.RoutineA routine assay calculates the concentration of endotoxin inunknowns by comparison to the performance of a series ofendotoxin standards. As part of a routine assay, the userhas the option to include a Positive Product Control (PPC) asa monitor for product inhibition or enhancement. A PPC is asample of product to which a known amount of endotoxinspike has been added. For quantitative assays, ourWinKQCL Software automatically calculates the amount ofendotoxin recovered in the PPC, allowing for a comparisonto the known amount of endotoxin spike.Inhibition/EnhancementThe Limulus Amebocyte Lysate reaction is enzymemediated and, as such, has an optimal pH range andspecific salt and divalent cation requirements. Occasionally,test samples may alter these optimal conditions to anextent that the lysate is rendered insensitive to endotoxin.Negative results <strong>with</strong> samples which inhibit the LAL test donot necessarily indicate the absence of endotoxin.An inhibition/enhancement assay is designed to determinewhat level of product dilution or other treatment overcomeinhibition or enhancement. Each product dilution must beaccompanied by a Positive Product Control (PPC). Forquantitative assays, our WinKQCL Software calculates theamount of endotoxin recovered in the PPC for comparison tothe known amount of endotoxin spike. In this manner it canbe determined which product dilutions are non-inhibitory.RSE/CSEAn RSE/CSE assay is designed to determine the potency ofa Control Standard Endotoxin (CSE) in terms of theconcentration units of the Reference Standard Endotoxin(RSE). The assay requires a single series of RSE dilutionsand one or more sets of dilutions of the CSE. If you buymatched reagents, we have already performed this test foryou. Our CSE is matched against the USP RSE.Matched CSE is either part of the kit or is available separately(for bulk kits).Initial QualificationAn Initial Qualification assay is required as part of thevalidation of the LAL assay and is also to be performed <strong>with</strong>each new lot of reagents. It serves to confirm reagentperformance and assure reproducibility. In addition, itshows analyst qualification. For this assay, a series ofendotoxin standards is prepared and tested in at leasttriplicate. To confirm sensitivity/linearity, the test resultmust meet regulatory requirements as defined bypharmacopeia. For gel clot assays, the determined end-pointmust fall between 2 λ and 0.5 λ of the labelled sensitivity.For the quantitative assays, the results are used to generatea standard curve which must have a correlation coefficientof ≥ |0.980|. The Initial Qualification assay does not providefor the inclusion of any samples.QC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com355
Overview of Endotoxin Detection MethodsEndotoxin Detection MethodsQualitative(Yes/No Answer)Product: PYROGENT Gel Clot LAL AssayQC Testing Solutions / Endotoxin Detection––Method – Visual inspection of gel formation––Maximum sensitivity – 0.03 EU/ml––Instrument required – A dry heat block or water bath■■Benefits––Simple LAL test not requiring sophisticatedinstrumentation and software––Less prone to interference from glucans8356North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Overview of Endotoxin Detection MethodsQuantitative(Results calculated from standard curve)Product: Kinetic-QCL Kinetic Chromogenic LAL Assay––Method – Kinetic measurement of color development––Maximum sensitivity – 0.005 EU/ml––Instrument required – Absorbance reader■■Benefits––Our most sensitive LAL-based method––Less sensitive to product inhibition––Ideal for biological products such as vaccines andantibioticsProduct: PYROGENT-5000 Kinetic Turbidimetric LALAssay––Method – Kinetic measurement of turbiditydevelopment––Maximum sensitivity – 0.01 EU/ml––Instrument required – Absorbance reader■■Benefits––Cost-effective method for water and large volumeparenteralsProduct: QCL-1000 Endpoint Chromogenic LAL Assay––Method – Endpoint measurement of color development––Maximum sensitivity – 0.1 EU/ml––Instrument required – Spectrophotometer orabsorbance reader■■Benefits––Results in 16 minutesProduct: PyroGene rFC Endpoint Fluorescent Assay––Method – Endpoint measurement of fluorescence––Maximum sensitivity – 0.005 EU/ml––Instrument required – Fluorescence reader■■Benefits––Elimination of false positive glucan reactions––Less lot-to-lot variability––Animal-free source and security of supply––FDA acknowledged alternative to LALQC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com357
Kinetic Chromogenic LAL Assay OverviewQC Testing Solutions / Endotoxin DetectionThe Kinetic-QCL Kinetic Chromogenic Assay is aquantitative, kinetic assay for the detection of Gramnegativebacterial endotoxin. A sample is mixed <strong>with</strong> thereconstituted LAL reagent in a 96-well plate and placed inan incubating absorbance plate reader that measuresabsorbance at 405 nm. The reaction is automaticallymonitored over time for the appearance of a yellow color.In the presence of endotoxin, the lysate will begin to cleavethe chromogenic substrate, causing the solution to becomeyellow. The time required for the change is inverselyproportional to the amount of endotoxin present. Theconcentration in unknown samples can be calculated froma standard curve. Due to the nature of this assay, theKinetic-QCL Assay is less impacted by inhibitory productsthat may interfere <strong>with</strong> the clotting mechanism inturbidimetric and gel clot assays. This feature, along <strong>with</strong>the sensitivity range of 0.005 to 50 EU/ml, makes thisassay optimal for biological products such as vaccines andantibiotics.Using our extensive experience and practical expertise <strong>with</strong>endotoxin detection and its regulatory requirements, Lonzahas developed an integrated system to support quantitativeendotoxin detection. Each system component has beenvalidated and can be verified. This all leads to reliable,reproducible and accurate quantitative results.Each quantitative system incorporates three elements:––Kinetic-QCL Kinetic Chromogenic LAL Assay––WinKQCL Endotoxin Detection and Analysis Software––Absorbance Plate Reader■■Benefits––Sensitivity range from 0.005 to 50 EU/ml■■Applications––Ideal for biological products such as vaccines andantibiotics■■Requirements––Incubating Absorbance Plate Reader––WinKQCL Software––LAL Reagent Water (for larger kits)––Pyrogen-free Test Tubes––LAL Reagent Grade Multi-well Plates8These elements integrate seamlessly to meet your testingrequirements, providing actionable results that allow you tobe confident in your critical decisions.358North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Kinetic-QCL Kinetic Chromogenic LAL AssayThe Kinetic-QCL Kinetic Chromogenic Assay kits containco-lyophilized lysate/substrate and matched controlstandard endotoxin (Cat. No. 50-650U also contains LALReagent Water).Bulk kit configurations are also available; kineticchromogenic LAL and matched control standard endotoxinare packaged separately but should be ordered together.These bulk configurations are made to order, and thereforerequire a lead time.Please contact Customer Service for more information.For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/kqcl2° C–8°C■■Benefits––Sensitivity range from 0.005 to 50 EU/ml––Less sensitive to product inhibition than assaysrequiring gel formation––Available in 192-, 2040- and 2400-test kit and bulkconfigurationsOrdering Information –Kinetic-QCL Kinetic Chromogenic LAL AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)50-650U 50-650U Kinetic-QCL Kinetic Chromogenic LAL Assay 8 × 24 tests/vial lysate, 2 vials endotoxin, 3 192 tests 0.005 to 50× 30 ml/vial LAL Reagent Water50-650NV 50-650NV Kinetic-QCL Kinetic Chromogenic LAL Assay 85 × 24 tests/vial lysate, 15 vials endotoxin 2,040 tests 0.005 to 5050-650H 50-650H Kinetic-QCL Kinetic Chromogenic LAL Assay 100 × 24 tests/vial lysate, 10 vials endotoxin 2,400 tests 0.005 to 50K50-643L K50-643L Kinetic-QCL Bulk Kinetic Chromogenic LAL Assay 25 × 24 tests/vial lysate 600 tests 0.005 to 50K50-643U K50-643U Kinetic-QCL Bulk Kinetic Chromogenic LAL Assay 100 × 24 tests/vial lysate 2,400 tests 0.005 to 50QC Testing Solutions / Endotoxin DetectionControl Standard Endotoxin for Kinetic-QCL Bulk Kinetic Chromogenic LALThe Control Standard Endotoxin, derived from E. coli O55:B5, is referenced against the USP Reference Standard Endotoxin.8Ordering Information – Control Standard Endotoxin for Kinetic-QCL Bulk Kinetic Chromogenic LALCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)E50-643L E50-643L Control Standard Endotoxin for Kinetic-QCL BulkKinetic Chromogenic LAL, E. coli Strain O55:B550 EU/ml 25 vials n/aRelated ProductsPageWinKQCL Endotoxin Detection and Analysis Software 378Incubating Absorbance Plate Reader 375LAL Reagent Grade Multi-well Plates 381LAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com359
QCL-1000 Endpoint Chromogenic LAL AssayThe QCL-1000 Endpoint Chromogenic LAL Assay is themost rapid of the LAL tests. This chromogenic LAL methodis based on the formation of a yellow color and is measuredphotometrically at 405–410 nm. With the QCL-1000 Assay,a multichannel pipette, a dry heat bath and a 96-well plate,you can run 96 reaction wells at one time. This assay canalso be run in tubes.For your convenience, Certificates of Analysis areavailable online:www.lonza.com/coaQC Testing Solutions / Endotoxin Detectionwww.lonza.com/qcl1000■■Benefits––Less sensitive to product inhibition than assaysrequiring gel formation––Sensitivity from 0.1 to 1 EU/ml––Quantitative results in 16 minutes––Flexible format -scalability 96-well plate or tubes, one ormultiple samples can be run at same time in one96-well plate––Can be run <strong>with</strong> simple spectrophotometer, no need forincubating absorbance plate reader■■Applications––Water testing––In-process testing––Final release testing––Testing plant-based material––Testing acidic/basic material2°C–8°COrdering Information – QCL-1000 Endpoint Chromogenic LAL AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)50-647U 50-647U QCL-1000 Endpoint Chromogenic LAL Assay 5 × 24 tests/vial lysate, 1 × 1 ml/vial endotoxin,2 × 6.5 ml/vial chromogenic substrate,2 × 30 ml/vial LAL Reagent Water50-648U 50-648U QCL-1000 Endpoint Chromogenic LAL Assay 5 × 60 tests/vial lysate, 2 × 1 ml/vial endotoxin,5 × 6.5 ml/vial chromogenic substrate120 tests 0.1 to 1300 tests 0.1 to 18Ordering Information – Control Standard Endotoxin for QCL-1000 Endpoint Chromogenic LALCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)E50-640 E50-640 Control Standard Endotoxin for QCL-1000Endpoint Chromogenic LAL, E. coli Strain0111:B4Endotoxin, 15 to 40 EU/ml 1 vial n/aRelated ProductsPageLAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380LAL Reagent Grade Multi-well Plates 381LAL Reagent Reservoirs 382Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382360North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Kinetic Turbidimetric LAL Assay OverviewThe PYROGENT-5000 Assay is a quantitative, kinetic assayfor the detection of Gram-negative bacterial endotoxin. Asample is mixed <strong>with</strong> the reconstituted LAL reagent in a96-well plate and placed in an incubating absorbance platereader that measures absorbance at 340 nm. The reactionis automatically monitored over time for the appearance ofturbidity.In the presence of endotoxin, the lysate will begin to gel,causing the solution to become cloudy or turbid. The timerequired for the change is inversely proportional to theamount of endotoxin present. The concentration in unknownsamples can be calculated from a standard curve.The PYROGENT-5000 Assay is perfect for laboratoriesneeding to process large numbers of samples. It is ideal forwater samples, large volume parenterals and water rinsefrom medical devices.Using our extensive experience and technical expertise <strong>with</strong>endotoxin detection and its regulatory requirements, Lonzahas developed an integrated system to support quantitativeendotoxin detection. Each system component has beenvalidated and can be verified. This all leads to reliable,reproducible and accurate quantitative results.Each quantitative system incorporates three elements:––PYROGENT-5000 Kinetic Turbidimetric LAL Assay––WinKQCL Endotoxin Detection and Analysis Software––Absorbance Plate ReaderThese elements integrate seamlessly to meet your testingrequirements, providing actionable results that allow you tobe confident in your critical decisions.■■Benefits––Sensitivity range from 0.01 to 100 EU/ml––Select from a wide range of kit sizes■■Applications––Cost-effective method for water and large volumeparenterals■■Requirements––Incubating Absorbance Plate Reader––WinKQCL Software––LAL Reagent Water (for larger kits)––Pyrogen-free Test Tubes––LAL Reagent Grade Multi-well PlatesQC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com361
PYROGENT-5000 Kinetic Turbidimetric LAL AssayPYROGENT-5000 kits contain turbidimetric lysate,reconstitution buffer for the lysate and matched controlstandard endotoxin. Bulk kit configurations are available;for those, kinetic turbidimetric LAL, reconstitution bufferand matched control standard endotoxin are packagedseparately but should be ordered together. These bulkconfigurations are made to order and therefore require alead time. Please contact Customer Service for moreinformation.QC Testing Solutions / Endotoxin Detection8For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/turb2°C–8°C■■Benefits––Sensitivity range from 0.01 to 100 EU/ml––Available in 100-, 200-, 2,250- and 4,500-test kit andbulk configurationsOrdering Information –PYROGENT-5000 Kinetic Turbidimetric LAL AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)N383 N383 PYROGENT-5000 Kinetic Turbidimetric LAL Assay 2 × 50 tests/vial lysate, 2 vials100 tests 0.01 to 100reconstitution buffer, 1 vial endotoxinN384 N384 PYROGENT-5000 Kinetic Turbidimetric LAL Assay 2 × 100 tests/vial lysate, 2 vials200 tests 0.01 to 100reconstitution buffer, 1 vial endotoxinN588 N588 PYROGENT-5000 Kinetic Turbidimetric LAL Assay 45 × 50 tests/vial lysate, 45 vials 2,250 tests 0.01 to 100reconstitution buffer, 10 vials endotoxinN688 N688 PYROGENT-5000 Kinetic Turbidimetric LAL Assay 45 × 100 tests/vial lysate, 45 vials 4,500 tests 0.01 to 100reconstitution buffer, 10 vials endotoxinT50-300L T50-300L PYROGENT-5000 Bulk Kinetic Turbidimetric LAL Assay 25 × 50 tests/vial lysate 1,250 tests 0.01 to 100T50-300U T50-300U PYROGENT-5000 Bulk Kinetic Turbidimetric LAL Assay 100 × 50 tests/vial lysate 5,000 tests 0.01 to 100T50-600L T50-600L PYROGENT-5000 Bulk Kinetic Turbidimetric LAL Assay 25 × 100 tests/vial lysate 2,500 tests 0.01 to 100T50-600U T50-600U PYROGENT-5000 Bulk Kinetic Turbidimetric LAL Assay 100 × 100 tests/vial lysate 10,000 tests 0.01 to 100Reconstitution Buffer for PYROGENT-5000 Bulk Kinetic Turbidimetric LALThe reconstitution buffer is provided for rehydration of the PYROGENT-5000 LAL Reagent.Ordering Information –PYROGENT-5000 Bulk Kinetic Turbidimetric Reconstitution BufferCat. No. NA Cat. No. EU Product Name Product Description SizeB50-300L B50-300L PYROGENT-5000 Bulk Kinetic Turbidimetric Reconstitution Buffer Reconstitution buffer for T50-300L 25 vialsB50-300U B50-300U PYROGENT-5000 Bulk Kinetic Turbidimetric Reconstitution Buffer Reconstitution buffer for T50-300U 100 vialsB50-600L B50-600L PYROGENT-5000 Bulk Kinetic Turbidimetric Reconstitution Buffer Reconstitution buffer for T50-600L 25 vialsB50-600U B50-600U PYROGENT-5000 Bulk Kinetic Turbidimetric Reconstitution Buffer Reconstitution buffer for T50-600U 100 vials362North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Control Standard Endotoxin for PYROGENT-5000 Bulk Kinetic Turbidimetric LALThe Control Standard Endotoxin, derived from E. coli O55:B5, is referenced against the USP Reference Standard Endotoxin.Ordering Information – Control Standard Endotoxin for PYROGENT-5000 Bulk Kinetic Turbidimetric LALCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity7460L 7460L Control Standard Endotoxin for PYROGENT-5000 Bulk KineticTurbidimetric LAL, E. coli Strain O55:B5100 EU/ml 25 vials n/aRelated ProductsPageWinKQCL Endotoxin Detection and Analysis Software 378Incubating Absorbance Plate Reader 374LAL Reagent Grade Multi-well Plates 381LAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380QC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com363
Alternative Method – PyroGene Recombinant Factor C AssayQC Testing Solutions / Endotoxin DetectionPyroGene Recombinant Factor C Assay is an animal-freealternative to LAL that has been accepted by the FDA as analternative method.* It is based on a recombinantlyproduced form of Factor C (rFC), the first component in thehorseshoe crab clotting cascade. It is activated by endotoxinbinding. The active moiety created then acts to cleave asynthetic substrate, which results in the release of afluorophore. The reaction is run in a 96-well microplate andmeasured at time zero and after a one-hour incubation in afluorescence microplate reader using excitation/emissionwavelengths of 380/440 nm.A global, multi-center study demonstrated that the recoveryof endotoxin from water and other tested products usingPyroGene was comparable to that of LAL-based methods.The results of the assay validation were published in thePharmacopeial Forum Vol. 36(1) (Jan. – Feb. 2010).In June 2012, the FDA issued the document “Guidance forIndustry Pyrogen and Endotoxins Testing: Questions andAnswers” which allows for the use of a recombinant Factor Cbased assay as an alternative to Limulus Amebocyte Lysate(LAL)-based assays.USP 28–NF 33 General Notices allows alternative methodsif they provide advantages regarding accuracy, sensitivity,precision, selectivity, or adaptability to automation.However, to use these alternative methods for final releasetesting, one may need to validate the test method on theirproducts as described in the general chapter "Validation ofCompendial Procedures” and it must be shown togive equivalent or better results.■■Benefits––Sensitivity range from 0.005 to 5 EU/ml––Higher endotoxin specificity––Elimination of false positive glucan reactions––Less lot-to-lot variability––Animal-free––Security of supply––FDA acknowledged alternative to LAL■■Applications––Water testing––In-process testing––Final release testing––Testing plant-based material■■Requirements––FLx800 Incubating Fluorescence Reader––WinKQCL Software––Pyrogen-free Test Tubes––LAL Reagent Grade Multi-well Plates––LAL Reagent Water (for larger kits)For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/pyrogene2°C–8°C8Each quantitative system incorporates three elements:––PyroGene Recombinant Factor C Assay––WinKQCL Endotoxin Detection and Analysis Software––Fluorescence Plate ReaderAvg Net Delta RFU0.005 0.05 0.5 510 000These elements integrate seamlessly to meet your testingrequirements, providing actionable results that allow you tobe confident in your critical decisions.1 0001000.01 0.1 1Standard curve illustrating assay range from 0.005 to 5 EU/ml* According to the FDA “Guidance for Industry - Pyrogen and Endotoxins Testing: Questions andAnswers” document from June 2012, alternative assays should be validated as described in theUSP General Chapter , “Validation of Compendial Procedures”.364North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Alternative Method – PyroGene Recombinant Factor C AssayContinuedComparison of Amplification Methods in Bacterial Endotoxin Detection Assays1970Detection stepcommon to allBET assaysSignal Amplification StepsMeasured CharacteristicGel C lotFactor CFactorBPro-clottingEnzymeClottingEnzymeCoagulogenCoagulinClot Formationvisual observationDevelopment TimelineCurrentKineticTurbidimetricEndpointChromogenicKineticChromogenicrFCFactorCFactorCFactorCFactorCFactorBFactorBFactorBFluorogenicSubstratePro-clottingEnzymePro-clottingEnzymePro-clottingEnzymeexcitation@ 380 nmClottingEnzymeClottingEnzymeClottingEnzymeFluorescence@ 440 nmDifference from previous methodsCoagulogenChromogenicsubstrateChromogenicsubstrateCoagulinDifference from previous methodsYellowColorYellowColorTurbidityMeasurement@340 nmColorMeasurement@405–410 nmColorMeasurement@405 nmLightMeasurement@440 nmrFC is the same binding protein operating in the LAL assay. The activated recombinant Factor C enzyme cleaves a substrate directly instead of activatinganother enzyme in a series (the LAL cascade). The substrate has a fluorescent tag, which gives a wide dynamic range <strong>with</strong> better resolution .Activity of 0.05 µg/ml LRM in EU/ml2.001.500,10Fluorescence (RFU)100,00010,0001,000QC Testing Solutions / Endotoxin Detection80.05100ChromogenicrFCGlucanGlucan + BlockerComparison of reactivity towards glucans between kinetic chromogenicLAL and rFC. The false positive signal from the LAL assay is reduced in thepresence of a glucan blocker. rFC does not detect any glucan activity, it isendotoxin-specific.100.01 0.10 1.00 10.00Concentration (EU/ml)Lot HL061M Lot HL076W Lot HL089KLot HL100W Lot HL131C Lot JL017ELot JL027ALot JL043EEndotoxin standard curves using 8 different lots of rFC. The log netfluorescence is proportional to the log endotoxin concentration and islinear in the 0.01–10 EU/ml range. Lot-to-lot standard curves exhibitexcellent reproducibility.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com365
PyroGene Validation TimelineA possible validation scheme is outlined below (onevalidation can be accomplished in as little as 5 daysassuming that the product has been previously validated<strong>with</strong> a quantitative LAL method). The validation scheme isidentical to that which would be needed for any LAL-basedmethod <strong>with</strong> just the addition of one extra step, "Validationof Alternative Method". Lonza offers a full validation protocolthat can be followed for your convenience. For furtherinformation please contact Scientific Support or your localsales representative.QC Testing Solutions / Endotoxin DetectionInhibition/Preparation Initial qualification enhancementValidation ofProduct validationtestingalternative method––IOPQ––Sensitivity assay for rFC––Initial qualification ofcurrent and rFC methodand analyst––Not necessary ifpreviously qualifiedreagent lots are used2 days 0.5 days >0.5 days(minimum)––Only for rFC––Time depending on extentof testing required to finda suitable dilution/pretreatment––Tests <strong>with</strong> both currentand rFC method––Good planning can savetime>1.5 days (minimum) 0.5 days––3 production lotsOrdering Information –PyroGene Recombinant Factor C Endpoint Fluorescent AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)50-658U 50-658U PyroGene Recombinant Factor CEndpoint Fluorescent Assay50-658NV 50-658NV PyroGene Recombinant Factor CEndpoint Fluorescent Assay2 × 96 tests/vial rFC enzyme solution, 2 × 6 ml/vialfluorogenic substrate, 2 × 5 ml/vial rFC assay buffer,2 vials endotoxin, 2 × 30 ml/vial LAL Reagent Water30 × 96 tests/vial rFC enzyme solution, 30 × 6 ml/vial fluorogenic substrate, 30 × 5 ml/vial rFC assaybuffer, 10 vials endotoxin192 tests 0.005 to 52,880 tests 0.005 to 58Related Products PagePyrogen-free Test Tubes 380LAL Reagent Grade Multi-well Plates 381LAL Reagent Reservoirs 382Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382366North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PYROGENT Gel Clot LAL Assay OverviewThe PYROGENT Gel Clot LAL Assay is a qualitative LAL testfor Gram-negative bacterial endotoxin. The gel clot assay isrun in tubes that are placed in a water bath or dry heat blockat 37ºC. After a one-hour incubation period, the tubes areinverted 180º. A firm clot that stays in the bottom of thetube indicates a positive reaction. If liquid flows down theside of the tube, the result is negative for endotoxin.Our unique production process of the PYROGENT Gel Clotlysate means that its sensitivity to (1,3)-β-D-glucans issignificantly reduced when compared to lysates usingalternative manufacturing methods. (1,3)-β-D-glucans arenon-endotoxin, non-pyrogenic substances found in yeast,fungi and cellulosic materials. These glucans can cause afalse positive result in the LAL test.Like other enzymatic reactions, the LAL assay is pHdependent. The PYROGENT lysate formulation contains abuffer to help <strong>with</strong> these adjustments. As a result, manyproducts will not require pH adjustments prior to testing.PYROGENT Gel Clot LAL kits are available in three formats:Lysate MatchedEndotoxinLiquidEndotoxinPYROGENT Gel Clot LAL ■PYROGENT Plus Gel Clot LAL ■ ■PYROGENT Ultra ■ ■■■Benefits––Easy-to-read qualitative results––Simple LAL test not requiring sophisticatedinstrumentation and software––Less susceptible to interference from glucans––Select from a wide range of kit sizes and sensitivities■■Applications––Water testing––In-process testing––Final release testing––Testing plant-based material––Testing acidic/basic material■■Requirements––A water bath or dry heat block––LAL Reagent Water (LRW)––Pyrogen-free Test TubesQC Testing Solutions / Endotoxin Detection8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com367
PYROGENT Gel Clot LAL AssayPYROGENT Gel Clot LAL Assay standard kit sizes include250 tests or 80 tests. Both the 250- and 80-test kits requiredepyrogenated 10 × 75 mm glass reaction tubes to run theassay.In addition, kits of PYROGENT Gel Clot LAL Single Test Vialsare available, which consist of 25 single use vials of lysate.Each single test vial serves as one reaction tube for anassay.These kits do not include a matched control standardendotoxin. However, the standard can be purchasedseparately (Control Standard Endotoxin, page 372).QC Testing Solutions / Endotoxin Detection8■■Benefits––Sensitivities of 0.03, 0.06, 0.125 and 0.25 EU/mlavailable––Easy-to-read qualitative results––Less susceptible to interference from glucans and pH––Also available as single test vials and as bulk kitsFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/gelclot2°C–8°COrdering Information – PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin)Cat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)N183-06 N183-06 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 16 tests/vial lysate 80 tests 0.06N183-125 N183-125 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 16 tests/vial lysate 80 tests 0.125N194-03 N194-03 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 50 tests/vial lysate 250 tests 0.03N194-06 N194-06 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 50 tests/vial lysate 250 tests 0.06N194-125 N194-125 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 50 tests/vial lysate 250 tests 0.125N184-25 N184-25 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 5 × 50 tests/vial lysate 250 tests 0.25N189-06 N189-06 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 25 single tests/vial lysate 25 vials 0.06single test vialsN189-125 N189-125 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin) 25 single tests/vial lysate 25 vials 0.125single test vialsN189-25 N189-25 PYROGENT Gel Clot LAL Assay (<strong>with</strong>out endotoxin)single test vials25 single tests/vial lysate 25 vials 0.25Related ProductsPageControl Standard Endotoxin for Gel Clot LAL 372Bulk kits 371LAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382368North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PYROGENT Plus Gel Clot LAL AssayThe PYROGENT Plus Gel Clot LAL Assay combinesPYROGENT LAL <strong>with</strong> a matched control standard endotoxintogether in one kit box. Standard kit sizes include 4,000tests, 200 tests or 64 tests. The 4,000-, 200- and 64-testkits require depyrogenated 10 × 75 mm glass reactiontubes to run the assay. In addition, kits of PYROGENT PlusGel Clot LAL Single Test Vials are available, which includes24 single test vials of lysate serving as the reaction tubes.For your convenience, the Certificate of Analysisdocumenting the FDA and USP required RSE/CSE correlationis available online:www.lonza.com/coawww.lonza.com/gelclot■■Benefits––Sensitivities of 0.03, 0.06, 0.125 and 0.25 EU/mlavailable––No need to purchase CSE separately––Also available as single test vials and as bulk kits2°C–8°COrdering Information – PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin)Cat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)N283-06 N283-06 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 16 tests/vial lysate, 1 vial endotoxin 64 tests 0.06N283-125 N283-125 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 16 tests/vial lysate, 1 vial endotoxin 64 tests 0.125N294-03 N294-03 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 50 tests/vial lysate, 1 vial endotoxin 200 tests 0.03N294-06 N294-06 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 50 tests/vial lysate, 1 vial endotoxin 200 tests 0.06N294-125 N294-125 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 50 tests/vial lysate, 1 vial endotoxin 200 tests 0.125N284-25 N284-25 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 4 × 50 tests/vial lysate, 1 vial endotoxin 200 tests 0.25N494-03 N494-03 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 80 × 50 tests/vial lysate, 20 vials endotoxin 4,000 tests 0.03N494-06 N494-06 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 80 × 50 tests/vial lysate, 20 vials endotoxin 4,000 tests 0.06N494-125 N494-125 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 80 × 50 tests/vial lysate, 20 vials endotoxin 4,000 tests 0.125N288-25 N288-25 PYROGENT Plus Gel Clot LAL Assay (<strong>with</strong> endotoxin) 80 × 50 tests/vial lysate, 20 vials endotoxin 4,000 tests 0.25N289-06 N289-06 PYROGENT Plus Gel Clot LAL Assay(<strong>with</strong> endotoxin), single test vials24 single tests/vial lysate, 1 × 1 ml vialendotoxin24 tests 0.06N289-125 N289-125 PYROGENT Plus Gel Clot LAL Assay(<strong>with</strong> endotoxin), single test vialsN289-25 N289-25 PYROGENT Plus Gel Clot LAL Assay(<strong>with</strong> endotoxin), single test vials24 single tests/vial lysate, 1 × 1 ml vialendotoxin,24 single tests/vial lysate, 1 × 1 ml vialendotoxin24 tests 0.12524 tests 0.25QC Testing Solutions / Endotoxin Detection8Related ProductsPageBulk kits 371LAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com369
PYROGENT Ultra Gel Clot LAL AssayThe PYROGENT Ultra Gel Clot LAL Assay contains 4 vials oflysate and a series of pre-mixed liquid endotoxin standardsthat are at the concentrations necessary for a standardcurve and positive product control (PPC). Because novortexing or dilutions are required, preparation time issubstantially reduced. Standard concentrations are 2 λ,1 λ, 0.5 λ, 0.25 λ, and 20 λ. Each kit is sufficient for200 tests.QC Testing Solutions / Endotoxin DetectionFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/gelclot■■Benefits––Eliminates the need for vortexing––Decreases preparation time––Simplifies the testing process––Increases productivity––Helps improve consistency of results2°C–8°COrdering Information – PYROGENT Ultra Gel Clot LAL AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)N594-03 N594-03 PYROGENT Ultra Gel Clot LAL Assay 5 × 5 ml gel clot liquid endotoxin standards(2 l, 1 l, 0.5 l, 0.25 l, 20 l), 4 × 50 tests/vial lysateN594-06 N594-06 PYROGENT Ultra Gel Clot LAL Assay 5 × 5 ml gel clot liquid endotoxin standards(2 l, 1 l, 0.5 l, 0.25 l, 20 l), 4 × 50 tests/vial lysateN594-125 N594-125 PYROGENT Ultra Gel Clot LAL Assay 5 × 5 ml gel clot liquid endotoxin standards(2 l, 1 l, 0.5 l, 0.25 l, 20 l), 4 × 50 tests/vial lysate200 tests 0.03200 tests 0.06200 tests 0.1258Related Products PagePyrogen-free Test Tubes 380Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382370North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
PYROGENT Bulk Gel Clot LAL AssayBulk kit configurations of PYROGENT Gel Clot LAL areavailable for laboratories using large volumes of reagents.These configurations are made to order and production leadtimes are required. Please inquire <strong>with</strong> your salesrepresentative for more information.■■Benefits––Bulk configurations for large volume use––Bulk kits <strong>with</strong> and <strong>with</strong>out endotoxin standard available––Sensitivities of 0.03, 0.06, 0.125 and 0.25 EU/mlavailableFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/gelclot2°C–8°COrdering Information –PYROGENT Bulk Gel Clot LAL AssayCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)E194L-03 E194L-03 PYROGENT Bulk Gel Clot LAL Assay 25 × 50 tests/vial lysate 1,250 tests 0.03E194L-06 E194L-06 PYROGENT Bulk Gel Clot LAL Assay 25 × 50 tests/vial lysate 1,250 tests 0.06E194L-125 E194L-125 PYROGENT Bulk Gel Clot LAL Assay 25 × 50 tests/vial lysate 1,250 tests 0.125E209L-25 E209L-25 PYROGENT Bulk Gel Clot LAL Assay 25 × 50 tests/vial lysate 1,250 tests 0.25F245U-06 F245U-06 PYROGENT Bulk Gel Clot LAL Assay 100 × 16 tests/vial lysate 1,600 tests 0.06F245U-125 F245U-125 PYROGENT Bulk Gel Clot LAL Assay 100 × 16 tests/vial lysate 1,600 tests 0.125E194U-03 E194U-03 PYROGENT Bulk Gel Clot LAL Assay 100 × 50 tests/vial lysate 5,000 tests 0.03E194U-06 E194U-06 PYROGENT Bulk Gel Clot LAL Assay 100 × 50 tests/vial lysate 5,000 tests 0.06E194U-125 E194U-125 PYROGENT Bulk Gel Clot LAL Assay 100 × 50 tests/vial lysate 5,000 tests 0.125E209U-25 E209U-25 PYROGENT Bulk Gel Clot LAL Assay 100 × 50 tests/vial lysate 5,000 tests 0.25QC Testing Solutions / Endotoxin Detection8Related Products PageLAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 382Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com371
Control Standard Endotoxin for PYROGENT Gel Clot LALLonza’s Control Standard Endotoxin is referenced againstthe USP Reference Standard Endotoxin.Certificates of Analysis showing potency are availableonline:www.lonza.com/coa2°C–8°CQC Testing Solutions / Endotoxin DetectionOrdering Information – Control Standard Endotoxin for PYROGENT Gel Clot LALCat. No. NA Cat. No. EU Product Name Product Description Size Sensitivity (EU/ml)50-506U 50-506U Inhibition Control for PYROGENT Gel Clot LAL Single Test Vials 5 vials n/aN186 N186 Control Standard Endotoxin for PYROGENT Gel Clot LAL Assays Endotoxin, E. coli O55:B5 5 vials n/a7360L 7360L Bulk Control Standard Endotoxin for PYROGENT Gel Clot LAL Assays Endotoxin, E. coli O55:B5 25 vials n/aRelated ProductsPageGel Clot LAL Assays 371LAL Reagent Water (LRW) 383Pyrogen-free Test Tubes 380Eppendorf® 2–200 µl Biopur® Pipette tips 382Eppendorf® 50–1000 µl Biopur® Pipette tips 3828372North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Instrumentation and SoftwareInstrumentation and SoftwareELx808 Absorbance Plate Reader 374FLx800 Fluorescence Plate Reader 375PyroTec Liquid Handling System 376WinKQCL Endotoxin Detection and Analysis Software 377QC Testing Solutions / Instrumentation & Software8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com373
ELx808 Absorbance Plate ReaderFor Kinetic-QCL and PYROGENT-5000 Kinetic LAL AssaysQC Testing Solutions / Instrumentation & Software8The ELx808 Absorbance Plate Reader has been validatedas part of our quantitative endotoxin detection systems.This 96-well reader features sample compartments <strong>with</strong>uniform temperature from well to well. It seamlesslyinterfaces <strong>with</strong> our WinKQCL Software.For the kinetic LAL assays, the ELx808 Reader is highlyreliable and easy to use. It features fully-automated readingof 96-well plates. This reader can also be used as a standardspectrophotometer and can read the results from theQCL-1000 Endpoint Chromogenic LAL Assay. It has a built-inmemory capability to store up to 55 assays, test results andstandard curves.On-Site service and preventive maintenance contracts areavailable to help ensure that your instrument is workingproperly.■■Benefits––Excellent temperature uniformity––Precise and accurate––Cost effective filter-based readerELx808 Absorbance Reader SpecificationsWavelength Range 340 to 900 nmFilters Supplied 340, 405, 450, 490 and 630 nmAbsorbance Range 0.000 to 4.000 Abs @ 400 to 900 nm0.000 to 3.000 Abs @ 340 to 400 nmTemperature Control 4°C above ambient to 50°CRead MethodKinetic or endpoint under WinKQCL ControlLight SourceTungsten Halogen BulbDimensions16-inches deep × 15.5-inches wide × 8.75-incheshigh (40.6 cm × 39.4 cm × 22.2 cm)Weight35 lb (15.9 kg)Ordering Information –ELx808 ReaderCat. No. NA Cat. No. EU Product Name Product Description25-315 BE25-315 ELx808 Reader Incubating Absorbance Reader25-515 Standard Kinetic System ELx808 Reader, data station <strong>with</strong> printer, WinKQCL Software25-342 25-342 Stepped Neutral Density Plate*7260522 7260522 BioTek® Universal Calibration Plate*05105 LAL3400508 Replacement Bulb for ELx808 Reader75053 Computer Connection Cable for ELx808 Reader00196004 00196004 UPS-APC 1500VA w/LCD Uninterruptible power supply00196005 00196005 4-Port Serial PCI Card Standard/low profile PCI card25-361 25-361 USB to Serial Port Converter*Recertification service is available (see Recertification Services, page347)374North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
FLx800 Fluorescence Plate ReaderFor PyroGene rFC Endpoint Fluorescent AssayThe FLx800 Reader is part of the quantitative endotoxindetection system that features the PyroGene rFC EndpointFluorescent Assay. This assay is measured <strong>with</strong> excitation/emission wavelengths of 380/440 nm. In addition tofluorescence measurements, this reader can be used as aluminometer. The FLx800 Fluorescence Plate Readerfeatures sample compartments <strong>with</strong> uniform temperaturefrom well to well. It seamlessly interfaces <strong>with</strong> our WinKQCLSoftware.On-Site service and preventive maintenance contracts areavailable to help ensure that your instrument is workingproperly.■■Benefits––Excellent temperature uniformity––Easy to useFLx800 Fluorescence Reader SpecificationsWavelength Range 300 to 700 nmTemperature Control 4°C above ambient to 50°CRead MethodEndpoint under WinKQCL ControlLight SourceTungsten Halogen BulbDimensions16-inches deep × 15-inches wide × 9-inches high(40.6 cm × 38.1 cm × 22.9 cm)Weight30 lb (13.6 kg)Ordering Information –FLx800 ReaderCat. No. NA Cat. No. EU Product Name Product Description25-344 25-344 FLx800 Reader Incubating Fluorescence ReaderBE200-108 Replacement Bulb for FLx800 Reader n/a00196004 00196004 UPS-APC 1500VA w/LCD Uninterruptible power supply00196005 00196005 4-Port Serial PCI Card Standard/low profile PCI card25-361 25-361 USB to Serial Port ConverterQC Testing Solutions / Instrumentation & Software8Related Products PageWinKQCL Endotoxin Detection and Analysis Software 378PyroGene rFC Endpoint Fluorescent Assay 366Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com375
PyroTec Liquid Handling SystemThe PyroTec Liquid Handling System is a roboticworkstation to help automate endotoxin detection testing.The system includes a user-defined platform size toaccommodate tips, reagent troughs and 96-well plates. Therobotic arm picks up tips and dispenses samples andreagents to 96-well plates. Heating units can incubateplates prior to delivery into plate readers.QC Testing Solutions / Instrumentation & SoftwareWinKQCL Software is fully integrated <strong>with</strong> the Tecan®Freedom EVOware® Software, allowing assay templates tobe executed using robotic scripts for the PyroTec LiquidHandling System.The PyroTec System can be tailored to your testing needs.Contact your local Lonza sales representative or office foradditional information.On-Site servicweand preventive maintenance contracts areavailable to help ensure that your instrument is workingproperly.■■Benefits––Flexible platform to automate filling of assay plates––To help high throughput labs manage their large dailysample requirementsOrdering Information – PyroTec Liquid Handling SystemCat. No. NA Cat. No. EU Product Name Product Description25-601 25-601 PyroTec 200 Liquid Handling System Robotic workstation for filling 96-well plates25-602 25-602 PyroTec 150 Liquid Handling System Robotic workstation for filling 96-well plates25-603 25-603 PyroTec 150 Positive ID System Robotic barcode scanner8Related ProductsPageWinKQCL Endotoxin Detection and Analysis Software 378Kinetic-QCL Kinetic Chromogenic LAL Assay 359PYROGENT-5000 Kinetic Turbidimetric LAL Assay 362PyroGene rFC Endpoint Fluorescent Assay 366376North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
WinKQCL Endotoxin Detection and Analysis SoftwareQuantitative methods such as the Kinetic-QCL Assaygenerate significant amounts of raw data that requirecareful analysis before reporting can take place. TheWinKQCL Software offers a fully integrated solution foryour quantitative endotoxin detection testing, datamanagement and reporting needs.WinKQCL 5 Software meets 21 CFR Part 11 technicalrequirements for electronic records and signatures, audittrails and database archiving. Reader validation tests canbe run from the WinKQCL Software and stored in the samedatabase. The built-in database backup and maintenancescheduler makes it easy to maintain the system.■■Benefits––Kinetic SmartStop monitoring feature to address splitpair, and other reaction conditions, <strong>with</strong>out sacrificingtime waiting for a fixed number of reads––Enterprise level IT features including wide area networksupport, ability to work across time zones, applicationvirtualization, Active Directory® integration and datasegregation by lab––Bi-directional interface <strong>with</strong> 3rd party databasesystems––Customizable endotoxin test reports––Multi-language user interface: English, French, German,Italian, Japanese, Spanish, Portuguese, SimplifiedChinese and Traditional Chinese■■Applications––For use <strong>with</strong> the ELx808 and FLx800 Readers.Extended reader integration now includes MolecularDevices® SpectraMax®, Gemini and VersaMaxReaders; and the BioTek® Eon and Synergy 2 Readers.The software is also interfaced <strong>with</strong> the Tecan® SunriseReader––Tecan® EVOware® interface integration for PyroTecLiquid Handling System––Supports all quantitative endotoxin detection assaysfrom Lonza including QCL-1000 Endpoint ChromogenicLAL, Kinetic-QCL Kinetic Chromogenic LAL,PYROGENT-5000 Kinetic Turbidimetric LAL andPyroGene rFC Endpoint Fluorescent Assays––Installation as a simple standalone system, or as aninterface <strong>with</strong> multiple robots and readers in multiplelabs around the world, all storing data in a singledatabaseThe user-friendly and flexible Template Manager allows youto customize plate layout <strong>with</strong> a click of a mouse using theSpeedFill and Drag n’ Drop features. The interactive andenhanced trending tools provide actionable results ondemand, helping you easily detect drift and enabling you tomake proactive decisions.The endotoxin detection instruments and software fromLonza are available fully supported <strong>with</strong> Installation,Operational and Performance Qualification (IOPQ) manualsand a WinKQCL 5 Software Validation Package. In addition,a trained specialist from Lonza can perform the IOPQ of thecomplete system to help you <strong>with</strong> your system validationprocess. Please inquire <strong>with</strong> your local sales representativefor further details.QC Testing Solutions / Instrumentation & Software8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com377
WinKQCL Endotoxin Detection and Analysis SoftwareContinuedOrdering Information – WinKQCL Endotoxin Detection and Analysis SoftwareCat. No. NA Cat. No. EU Product Name Product Description25-501 25-501 WinKQCL 5 Software Package Installation Disc, Workgroup License, Reader License25-502 25-502 WinKQCL 5 Workgroup License Additional Workgroup License25-503 25-503 WinKQCL 5 Reader License Additional Reader License25-504 25-504 WinKQCL 5 Qualification Manual IOPQ manual for software and readers25-505 25-505 WinKQCL 5 Validation Package Disc containing software validation information25-339S 25-339S System Qualification Service IQ/OQ/PQ validation on site, labour onlyQC Testing Solutions / Instrumentation & SoftwareRelated ProductsPageKinetic-QCL Kinetic Chromogenic LAL Assay 359PYROGENT-5000 Kinetic Turbidimetric LAL Assay 362PyroGene rFC Endpoint Fluorescent Assay 366ELx808 Incubating Absorbance Reader 374FLx800 Incubating Fluorescence Reader 3758378North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Accessory ProductsAccessory ProductsIntroduction380Test Tubes 380Sample Containers 381Plates381Pipette Tips 382Reservoirs382Dry Heat Block, Inserts and Vortex Mixer 383LAL Reagent Water 383β-G-Blocker384PYROSPERSE384MgCl 2 385Tris Buffer 385Endotoxin and Endotoxin Challenge Vials 386QC Testing Solutions / Accessory Products8Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com379
IntroductionIn addition to the endotoxin detection assay kits,instruments and software, Lonza offers many of theaccessory items necessary to run endotoxin detectionassays. Many of the items have been tested <strong>with</strong> theKinetic-QCL Kinetic Chromogenic LAL Assay to help ensuretheir compatibility <strong>with</strong> our endotoxin detection methods.We also offer products such as the Endotoxin ChallengeVials to help <strong>with</strong> your oven depyrogenation validations.QC Testing Solutions / Accessory Products8Test TubesAll test tubes are made from USP Type I flint borosilicateglass.Both N201 and N205 are recommended for use as reactiontubes in gel clot assays. N201 are provided <strong>with</strong>polypropylene screw caps. Product number N207 isrecommended for dilution of endotoxin standards and testsamples for all endotoxin detection assays.■■Benefits––Certified to contain less than 0.005 EU/ml endotoxinFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/accessoriesOrdering Information – Test TubesCat. No. NA Cat. No. EU Product Name Product Description SizeN207 N207 Pyrogen-free Test Tubes Without caps, 13 × 100 mm 30/foil packN201 N201 Pyrogen-free Test Tubes With caps, 10 × 75 mm 50/boxN205 N205 Pyrogen-free Test Tubes Without caps, 10 × 75 mm 50/foil pack380North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Sample ContainersSample containers are meant for transporting productsamples for endotoxin analysis or sample storage. Propercontainer and storage conditions need to be validated foreach individual sample.Products 80-507L and 80-507U contain 10 ml glass vials<strong>with</strong> screw caps. Products BE2098 and BE2099 are plasticsample containers which offer greater capacity at a reducedcost.■■Benefits––Certified to contain less than 0.005 EU/ml endotoxinFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/accessoriesOrdering Information – Sample ContainerCat. No. NA Cat. No. EU Product Name Product Description Size80-507L 80-507L Sample Containers Depyrogenated, 10 ml glass bottle <strong>with</strong> screw cap 25/box80-507U 80-507U Sample Containers Depyrogenated, 10 ml glass bottle <strong>with</strong> screw cap 100/boxBE2098 Polypropylene Sample Containers Endotoxin tested, 50 ml tubes 50/packBE2099 Polystyrene Sample Containers Endotoxin tested, 15 ml tubes 50/packPlatesQC Testing Solutions / Accessory ProductsThese 96-well plates can be used <strong>with</strong> the QCL-1000Endpoint Chromogenic LAL Assay, Kinetic-QCL KineticChromogenic LAL Assay, PYROGENT-5000 KineticTurbidimetric LAL Assay and PyroGene rFC EndpointFluorescent Assay. Each case contains individually wrappedplates.■■Benefits––Certified to contain less than 0.0005 EU/well endotoxinFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/accessories8Ordering Information – PlatesCat. No. NA Cat. No. EU Product Name Product Description Size25-340 BE25-340 LAL Reagent Grade Multi-well Plates 96-well plates, endotoxin-tested (
Pipette TipsPyrogen-free pipette tips are to be used when testing <strong>with</strong>any of our endotoxin detection systems.Eppendorf® Biopur® pipette tips are certified to contain
Dry Heat Block, Inserts and Vortex MixerThe dry heat block is used for incubation of LAL gel clotassays.The aluminum block <strong>with</strong> lid adaptor for a dry heat blockallows a 96-well plate to be incubated at 37°C for use <strong>with</strong>QCL-1000 Endpoint Chromogenic LAL Assay.www.lonza.com/accessoriesOrdering Information – Dry Heat Block, Inserts and Vortex MixerCat. No. NA Cat. No. EU Product Name Product Description Size25-038 Aluminum Block <strong>with</strong> Lid Heat block insert for 96-well plate EachBEF3503 Aluminum Insert Block for Techne Dry Heat Block For 20 tubesBEDB-2D Techne Dry Heat Block Digital, from 25°C to 100°C, requires 2 × BEF3503 For 40 tubesFDB03DD Techne Dry Heat Block Digital, from 25°C to 100°C, requires 3 × BEF3503 For 60 tubesBENP5051 Vortex Genie® 2LAL Reagent WaterLAL Reagent Water is recommended for reconstituting LALreagents, as well as the dilution of control standardendotoxin and test samples for endotoxin testing. LALReagent Water is equivalent to Water for Bacterial EndotoxinsTest (BET).QC Testing Solutions / Accessory ProductsFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coa8www.lonza.com/accessories■■Benefits––Certified to contain less than 0.005 EU/ml endotoxin––Available in a variety of sizes2°C–8°C (W50-640)15°C–30°C (W50-100, W50-500, W50-1000)Ordering Information – LAL Reagent WaterCat. No. NA Cat. No. EU Product Name Product Description SizeW50-640 W50-640 LAL Reagent Water
β-G-Blocker(1,3)-β-D-glucans can produce false positive results in LALassays. Some examples of glucan sources include yeastand cellulosic materials including hemodialysis filters. Ourβ-G-Blocker may be used <strong>with</strong> any of our LAL assays.NOTE: PYROGENT Gel Clot LAL products typically do notrequire β-G-Blocker.For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coaQC Testing Solutions / Accessory Products8www.lonza.com/accessories■■Benefits––Certified to contain less than 0.005 EU/ml endotoxin––Functionality tested to demonstrate a reduction ofenhancement caused by (1,3)-β-D-glucansPYROSPERSEPYROSPERSE, a dispersing agent, helps eliminateendotoxin binding or masking in some samples – solvingproblems of inhibitory behavior. Examples of samples thatmay show endotoxin binding behavior include plasmaprotein fractions, electrolyte solutions, and lipid emulsions.PYROSPERSE may be used <strong>with</strong> any of our LAL kits.For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coa2°C–8°COrdering Information – β-G-BlockerCat. No. NA Cat. No. EU Product Name Product Description SizeN190 N190 β-G-Blocker Glucan blocker, 5 ml/vial 5 vialswww.lonza.com/accessories■■Benefits––Endotoxin and functionality tested2°C–30°C (unopened)Ordering Information –PYROSPERSE Dispersing AgentCat. No. NA Cat. No. EU Product Name Product Description SizeN188 N188 PYROSPERSE Dispersing Agent 5 ml/vial 5 vials384North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
MgCl 2MgCl 2 can be used as the sample diluent when attemptingto overcome inhibitory chelation effects. Examples ofsamples which chelate divalent cations include heparin andEDTA. MgCl 2 may be used to prepare samples for anyendotoxin detection assay.For your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/accessories■■Benefits––Certified to contain less than 0.005 EU/ml endotoxin2°C–30°C (unopened)Ordering Information – MgCl 2 10 mM SolutionCat. No. NA Cat. No. EU Product Name Product Description SizeS50-641 S50-641 MgCl 2 10 mM Solution 30 ml/vial 1 vialTris BufferTris Buffer can be used in place of water as the samplediluent for highly acidic or basic samples (for endotoxintesting, samples and sample dilutions should be betweenpH 6–8 after lysate addition). Tris Buffer may be used toprepare samples for any of our endotoxin detection assays.QC Testing Solutions / Accessory ProductsFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coa8www.lonza.com/accessories■■Benefits––Certified to contain less than 0.005 EU/ml endotoxin2°C–8°COrdering Information – Tris Buffer 50 mMCat. No. NA Cat. No. EU Product Name Product Description SizeS50-642 S50-642 Tris Buffer 50 mM 30 ml/vial 1 vialEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com385
Endotoxin and Endotoxin Challenge VialsEndotoxin (E. coli) Challenge Vials are for use in ovenvalidation studies. Each vial contains >1,000 EU/vial. Thevials may be tested using any of our endotoxin detectionkits. Product 00193783 contains high potency endotoxinand is intended for use in endotoxin removal systemchallenges, i.e. depyrogenation ovens, and other spikingstudies. Each vial contains >1,250,000 EU/vial. E700 is theUSP Reference Standard Endotoxin. Each vial contains10,000 EU/vial.QC Testing Solutions / Accessory ProductsFor your convenience, Certificates of Analysis are availableonline:www.lonza.com/coawww.lonza.com/accessories■■Benefits––Products 00192568 and 00193783 are devoid of fillers,which complies <strong>with</strong> the USP Endotoxin Indicatorsmonograph2°C–8°COrdering Information – Endotoxin and Endotoxin Challenge VialsCat. No. NA Cat. No. EU Product Name Product Description Size00193783 00193783 Endotoxin > 1,25 million EU/vial 5 vials00192568 00192568 Endotoxin Challenge Vials >1,000 EU/vial 25 vialsE700 E700 USP Reference Standard Endotoxin 10,000 EU/vial 1 vial8386North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Bio<strong>Research</strong>9 Technical Information9Primary Cell Culture and Media 391Primary Cell Methods 404Transfection413Media, Reagents and Sera 431Electrophoresis and Analysis 456
Technical InformationTechnical Information9Introduction390Primary Cell Culture and MediaOverview of Cell Culture Process forClonetics Human and Animal Cells* 391Safety Precautions <strong>with</strong> Clonetics Cells 392Media Preparation 392Clonetics and Poietics Cell Culture Media 394Pulmonary Epithelial Cell Media Options 395Endothelial Cell Media Options 396Fibroblast Cell Media Options 397Hepatocyte Media 397Keratinocyte Cell Media Options 398Mammary Epithelial Cell Media, Serum-free 399Melanocyte Cell Medium, Serum-free 399Neural Cell Medium, Low Serum 399Sertoli Cell Medium 399Prostate Epithelial Cell Medium, Serum-free 400Renal Cell Media, Low Serum 400Retinal Pigment Epithelial Cell Media 400Primary Neuron Growth Medium, Serum-free 400Human Mesenchymal Stem Cell Media 401Rat Mesenchymal Stem Cell Media 401Human Adipose-Derived Stem Cells 402Preadipocyte Growth Medium 402Osteoclast Growth Medium 402Neural Progenitor Growth Medium 402Skeletal Cell Medium 403Skeletal Muscle Cell Medium 403Smooth Muscle Cell Medium 403Stromal Cell Medium, Low Serum 403Primary Cell MethodsCulture Set-up – Adherent Cell Types 405Thawing Cells – Adherent Cell Types 405Seeding – Adherent Cell Types 406Proliferating Cells – Adherent Cell Types 406Subculturing – Adherent Cell Types 407Subculturing into 96-well Plates 409Instructions for Cryopreservation 410Improving Cell Yield and Viability During Subculture 411Custom Cell Isolation Services 412TransfectionCell Culture Tips for Cell Lines and Primary CellsPrior to Transfection 413Important Vector Factors for Gene Expression 415Essentials for Preparing a Transfection Experiment<strong>with</strong> Plasmid DNA 419Guideline for Generation of Stable Cell Lines 420Designing an RNAi Experiment Using Nucleofection 427Media, Reagents and SeraCell Culture Technical Information 431Adaptation of Cell Cultures to Serum-free Medium 431Cryopreservation and Reconstitution 433Determination of Cell Numbers 434Powdered Media Preparation 436Subculturing Procedures for Mammalian Cells 437Formulations for BioWhittaker Classical Media 439Cell Culture Reagent Formulations 455Electrophoresis and AnalysisFrequently Asked Questions – Protein Analysis 458Agarose Types 460Preparation of Agarose Gels 461Loading Buffers 466Detection and Sizing of DNA in Agarose Gels 467Detecting DNA <strong>with</strong> GelStar®, SYBR® Green I or IINucleic Acid Gel Stains 470Detecting DNA <strong>with</strong> Ethidium Bromide 472Recovery of DNA from Agarose Gels 473Protein Separation in Polyacrylamide Gels 475Blotting Proteins from Polyacrylamide Gels 477Electrophoretic Theory 478Safety and Environmental Precautions 479Specific Chemical Hazards 479388North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Technical InformationTechnical InformationPrimary Cell Culture and Media 391Primary Cell Methods 404Transfection413Media, Reagents and Sera 431Electrophoresis and Analysis 4569Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com389
IntroductionTechnical InformationWe continue to set the standard for quality in the researchindustry <strong>with</strong> our long-established and trusted brands forprimary cells, media, transfection and separations. We alsostrive to lead the industry in Scientific Support for ourcustomers. In addition to our global Scientific Support Team,we offer a broad selection of support tools for a variety ofapplications from basic discovery to applied research. Thefollowing chapter provides a selection of technical tips andguidelines to support you research, and offers just ahighlight of the large body of support materials available atour website.Any technical advice or guidance furnished orrecommendation made by Lonza set forth herein is providedin good faith, but Lonza makes no warranty, either expressor implied, as to its completeness or accuracy or the resultsto be obtained from use thereof. Any questions should bedirected to Lonza at the contact information set on thebottom of this catalog.Primary Cells and MediaWhether you are using primary cells or cell lines in yourresearch, we have product solutions and technical supportmaterials to make your cell culture succeed. In addition togeneral cell culture workflow guidelines and mediapreparation instructions, we provide classical mediaformulations, thawing and set-up protocols, and instructionsfor serum-free media weaning.TransfectionIn this chapter we include the most critical guidelines forpreparing cells for viable, high effeciency transfection. Inaddtition to tips on substrate preparation and cell handling,we provide guidelines for successful siRNA experimentsand generation of stable clones. For even further detail, ourwebsite offers a large collection of bench guides and whitepapers, created by our Scientific Support Team, discussingimportant considerations for successfull transfection.www.lonza.com/technical-libraryElectrophoresis and AnalysisFrom detailed specifications on agarose and size markers,to specific instructions for DNA recovery and WesternBlotting of proteins, we cover the basics needed to ensuresuccessful separation, detection and analysis of nucleicacids and proteins in agarose and polyacrylamide gels. Thissection provides just a fraction of the comprehensiveinformation available in our online Sourcebook forElectrophoresis.www.lonza.com/sourcebook9390North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Primary Cell Culture and MediaOverview of Cell Culture Process for Clonetics Human and Animal Cells*Storage Requirements:CellsUpon arrival, immediately removecryo preserved cells from dry ice andplace immediately into liquid nitrogen.If no dry ice is left in the package, thawcells, immediately place them intoculture vessels and call your ScientificSupport Specialist.MediumStore Clonetics Cell Culture Mediumin a 4°C refrigerator. When usingmedium, under sterile conditions, takethe amount you need and returnbottle to the refrigerator. Always bringmedium to room temperature beforeuse.Supplements and ReagentsIf you to plan to subculture <strong>with</strong>inthree days, store all growthsupplements, HEPES Buffered SalineSolution and Trypsin NeutralizingSolution at 4°C. Trypsin/EDTA Solutionhas a limited shelf life at 4°C. If, uponarrival , Trypsin/EDTA is thawed,immediately aliquot and refreeze at-20°C. If Trypsin/EDTA is frozen, storeat -20°C. If you do not plan to set upthe cell culture <strong>with</strong>in 3 days, store allgrowth supplements and subculturereagents in a -20°C freezer.1–2DaysBulletKit Medium▼Add SingleQuots KitProliferating Cultures▼Incubate 3–4 hoursReplace medium and incubateUnpack and store cells▼Prepare medium▼Prepare cells▼Maintenance: Every 40 hourschange medium, increase asnecessaryWhen at 60–90% confluence,subcultureAssess cell yield and viability,optimize as necessaryMaintenance: After 24 hours,change mediumSupplemented Media▼Add BPE or BBECryopreserved Cultures▼Calculate number of flasks/chambers/dishes requiredAdd medium to culturevessel(s) and incubate for atleast 30 minutesThaw cells in water bathResuspend cells in cryovialDispense equal amounts ofcells into flask5–9DaysTechnical Information / Primary Cell Culture and Media9Incubate and re-examine culture*Exceptions: NHEM, rat and mouse neural cells, rat cardiac myocytes,h NHEPS, sm NHEPS, rt NHEPS, and bMVEC-BEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com391
Safety Precautions <strong>with</strong> Clonetics CellsTechnical Information / Primary Cell Culture and Media9––As a precaution against contamination, follow allprocedures for handling products of human originoutlined in Biosafety in Microbiological and BiomedicalLaboratories (BMBL), 5th Edition (see link below).––Always wear gloves and safety glasses when working<strong>with</strong> all materials. Exercise caution when working <strong>with</strong>cryopreserved cells; rapid temperature changes maycause splattering of liquid nitrogen.––Wash hands thoroughly after performing all procedures.––Do not smoke, eat or drink in areas where reagents orcells are handled.––Never pipette by mouth.––Products of human and animal origin are potentialbiohazards. Although most provided human cells aretested and confirmed negative for HIV-1, Hepatitis B andC, proper precautions must be taken to avoid inadvertentexposure.Media PreparationBefore You BeginPerform the following steps before you begin media or cellpreparation:1. Prepare a sterile field.––A sterile field consists of a Class II biological safetycabinet <strong>with</strong> a front access opening and filteredlaminar airflow, or an equivalent device2. Determine the amount of medium required:––Review the Growth Area of Common Plasticwaretable (see page 377)to determine the amount ofmedium to be used3. Sterile instruments and vessels required:––Sterile disposable serological pipettes––Micropipettes and sterile pipette tips––Adjustable multichannel pipette or repeating pipette––Sterile reservoirs for use <strong>with</strong> multichannel pipette––Sterile 15 ml centrifuge tubes––Cell culture flasks, or multiwell, flat-bottom tissueculture plates––Hemacytometer or cell counterwww.cdc.gov/biosafety/publications/bmbl5/Caution: Clonetics and Poietics Products containhuman sourced material. Treat as potentiallyinfectious.4. Other required supplies:––70% alcohol (ethanol or isopropanol)––Growth medium (cell type specific)––Protective gloves and garments––Trypan Blue5. Plan and prepare for initial set up:––Base the set-up on the number of cells indicated onthe Certificate of Analysis accompanying theproduct6. Check the calibration on the humidified incubator.Incubator should be humidified and set to 5% CO 2, 95%air, and 37°C.392North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Media PreparationContinuedPerform the Following Steps in a Sterile FieldFor a bottle of fully supplemented medium, do the following:1. Add Bovine Brain Extract (BBE) or Bovine PituitaryExtract (BPE), if required, to a 500 ml bottle of basalmedium.2. Detach the BBE or BPE supplement from the mediumbottle.3. Decontaminate the vial and medium bottle <strong>with</strong> ethanolor isopropanol.4. Add the entire contents of the vial (approximately 2 ml)to the medium <strong>with</strong> a pipette; rinse the vial <strong>with</strong> mediumand pipette the contents back into the 500 ml bottle.5. Replace the cap and swirl the medium gently a fewtimes to mix.6. Record the date the BBE or BPE was added on themedium label. A fully reconstituted Complete Mediashould be used <strong>with</strong>in 30 days; this supplementedmedium will now be referred to as a growth medium.For a BulletKit Medium Medium, Perform the FollowingSteps1. Decontaminate the external surfaces of theSingleQuots Cryovials and the basal media bottle <strong>with</strong>ethanol or isopropanol.2. Aseptically open each cryovial and add the entireamount to the basal medium <strong>with</strong> a pipette.3. Rinse each cryovial <strong>with</strong> the medium. It may not bepossible to recover the entire volume listed for eachcryovial; small losses, even up to 10%, should not affectthe cell growth characteristics of the supplementedmedium.4. Transfer the label provided <strong>with</strong> each kit to the basalmedium bottle being supplemented. Use it to record thedate and amount of each supplement added; werecommend that you place the completed label over thebasal medium label to avoid confusion or possibledouble supplementation.5. Record the new expiration date on the label based onthe shelf life. A fully reconstituted BulletKit MediumMedium should be used <strong>with</strong>in 30 days; thissupplemented medium will now be referred to as agrowth medium.NOTE: If there is concern that sterility was compromised during thesupplementation process, the entire newly prepared growth mediummay be re-filtered to assure sterility. If you re-filter, use a sterile0.2-micron filter. Routine re-filtration is not recommended as someprotein loss may occur <strong>with</strong> each filtration.Technical Information / Primary Cell Culture and MediaGrowth Area of Common PlasticwareFlasksEffectiveGrowth AreaCell CultureMedium RequiredInitial Numberof Cells to Seedat 2,500 cells/cm 2Initial Numberof Cells to Seedat 3,500 cells/cm 2T-25 25 cm 2 5 ml 62,500 87,500 125,000T-75 75 cm 2 15 ml 187,500 262,500 375,000T-150 150 cm 2 30 ml 375,000 525,000 750,000Initial Numberof Cells to Seedat 5,000 cells/cm 2DishesEffectiveGrowth AreaCell CultureMedium RequiredInitial Numberof Cells to Seedat 2,500 cells/cm 2Initial Numberof Cells to Seedat 3,500 cells/cm 235 mm 9.6 cm 2 2 ml 20,000 28,000 40,00060 mm 21 cm 2 5 ml 52,500 73,500 105,000100 mm 55 cm 2 11 ml 137,500 192,500 275,000150 mm 148 cm 2 30 ml 370,000 518,000 740,000Initial Numberof Cells to Seedat 5,000 cells/cm 29MultiwellPlatesEffectiveGrowth Areaper wellCell CultureMedium Requiredper well/total6-well 9.60 cm 2 2 ml / 12 ml 96,00012-well 3.80 cm 2 1 ml / 12 ml 38,00024-well 2.00 cm 2 0.5 ml / 12 ml 20,00048-well 0.75 cm 2 150 µl / 7 ml 7,50096-well 0.32 cm 2 100 µl / 10 ml 3,200Initial Numberof Cells to Seedat 10,000 cells/cm 2Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com393
Clonetics and Poietics Cell Culture MediaTechnical Information / Primary Cell Culture and MediaMedium SpecificationsMedium is formulated for optimal growth of specific typesof primary cells. It can be purchased as basal medium<strong>with</strong>out supplements, as fully supplemented growthmedium, or in a conveniently packaged BulletKit Mediumthat allows user control over supplement type andconcentration. Each type of medium is tested for its abilityto support growth or differentiation of the intended primarycell. Biochemical and sterility tests are also performed onevery lot of medium. Certificates of Analysis are availableupon request for all medium products.Basal MediumBasal medium has been optimized for specific types ofprimary cells. Basal medium does not contain growthfactors necessary for propagation of cells. Growth factorsmust be added to enhance plating efficiency and cellularproliferation. Optimized formulations make it possible toperform research on a wide variety of primary cell types.Results of years of media development are available to you foruse in your own research.Complete MediumComplete medium is fully supplemented growth medium(BPE and BBE are packaged separately) and contains all ofthe growth factors and supplements necessary for thepropagation of specific types of primary cells in culture.Undefined supplements are avoided when possible and usedonly at minimal levels when necessary. Standardformulations of all growth media include antimicrobials.Antimicrobial-free media are available as a special order.BulletKit MediumA BulletKit Medium provides flexibility in final mediumformulation and increased shelf life. Each BulletKitMedium contains basal medium and pre-measured,single-use aliquots (SingleQuots Kit) of growth factorsand antimicrobial agents to formulate the fullysupplemented growth medium of your choice.9394North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Pulmonary Epithelial Cell Media OptionsPulmonary Epithelial cell media are serum-free media thathave been optimized for the proliferation and differentiationof certain cells. Each component of the basal medium andeach growth supplement are carefully titered for optimalgrowth by our R&D team. We offer many media choices forthe growth of airway cells, allowing for desired performanceand formulation flexibility. When selecting a medium to use,refer to specific media recommendations or call ScientificSupport for assistance.Product InformationCat. No. Product DescriptionBEGM BulletKit Medium––Best growth of NHBE and DHBE – Normal and DiseasedHuman Bronchial/Tracheal Epithelial Cells in mediumcontaining antimicrobialsSAGM BulletKit Medium––Superior growth for SAEC and D-SAEC – Normal andDiseased Human Small Airway Epithelial CellsB-ALI BulletKit Medium––Differentiation of Bronchial/Tracheal epithelial Cells inan Air Liquid Interface cultureCC-3170 BEGM BulletKit Medium Bronchial Epithelial Cell Growth Medium BulletKit, Serum-freeCC-3171 BEBM Basal Medium Bronchial Epithelial Cell Basal Medium, Serum-freeCC-4175 BEGM SingleQuots Kit Formulates BEBM to BEGM, BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml;Transferrin, 0.5 ml; Insulin, 0.5 ml; Triiodothyronine, 0.5 ml; GA-1000, 0.5 ml; Retinoic Acid, 0.5 mlCC-3118 SAGM BulletKit Medium Small Airway Epithelial Cell Growth Medium BulletKit, Serum-freeCC-3119 SABM Basal Medium Small Airway Epithelial Cell Basal Medium, Serum-freeCC-4124 SAGM SingleQuots Kit Formulates SABM to SAGM, BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml;Transferrin, 0.5 ml; Insulin, 0.5 ml; Triiodothyronine, 0.5 ml; GA-1000, 0.5 ml; Retinoic Acid, 0.5 ml; BSA-FAF, 5.0 ml00193514 B-ALI BulletKit Medium Bronchial Air Liquid Interface BulletKit, Serum-freeThe B-ALI BulletKit Medium includes a 250 ml bottle of B-ALI Growth Basal Medium,a 500 ml bottle of B-ALI Differentiation Basal Medium, and a B-ALI SingleQuots Kit00193515 B-ALI SingleQuots Kit Formulates B-ALI basal media to growth and differentiation media, Transferrin, 0.9 ml; B-ALI-Inducer, 1ml; BPE, 3.3 ml; Epinephrine, 0.9 ml; GA-1000, 0.9 ml; hEGF, 0.9 ml; Hydrocortisone, 0.9 ml; Insulin, 0.9ml; Retinoic Acid, 0.9 ml; Thiodothyronine, 0.9 ml00193516 B-ALI Growth Basal Medium Bronchial Air Liquid Interface Basal Medium Growth, Serum-free00193517 B-ALI DifferentiationBasal MediumBronchial Air Liquid Interface Basal Medium Differentiation, Serum-freeTechnical Information / Primary Cell Culture and Media9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com395
Endothelial Cell Media OptionsTechnical Information / Primary Cell Culture and MediaEndothelial cell media are low serum media optimized forthe proliferation of endothelial cells. Each component of thebasal medium and each growth supplement is carefullytitered for optimal growth by our R&D team. We currentlyoffer four Clonetics Media for the growth of endothelialcells allowing for desired performance and formulationflexibility. When selecting a medium to use, refer to specificmedium recommendations or call Scientific Support forassistance.EGM and EGM BulletKit Medium––Basal medium developed for normal human endothelialcells in a low-serum environment––EGM Complete Medium is supplemented growthmedium and includes an attached aliquot of BovineBrain Extract (BBE)––EGM BulletKit Medium includes basal medium <strong>with</strong>supplements and growth factors in separate, frozenaliquots––Final serum concentration is 2%––EGM Media can be used to grow all of CloneticsEndothelial Cells except microvascular, coronary arteryand iliac arteryEGM MV BulletKit Medium––Developed for microvascular and coronary arteryendothelial cells––Same basal medium as in EGM Media––Final serum concentration is 5%EGM-2 BulletKit Medium––Refinements to basal medium and the growth factors––Does not contain BBE––Final serum concentration is 2%––Improved cell proliferation over EGM––EGM-2 can be used to grow all of Clonetics EndothelialCells except microvascular, coronary artery and iliacarteryEGM-2MV BulletKit Medium––Developed for the enhanced growth of microvascularendothelial cells––Does not contain BBE––Final serum concentration increased to 5%9Product InformationCat. No. Product DescriptionCC-3125 EGM MV BulletKit Medium Microvascular Endothelial Cell Growth Medium BulletKit Medium w/ 5% FBSCC-3121 EBM Basal Medium Endothelial Cell Basal Medium, Serum-freeCC-4143 EGM MV SingleQuots Kit Formulates EBM to EGM MV; BBE, 2 ml; hEGF, 0.5 ml; Hydrocortisone, 0.5 ml; FBS, 25 ml; GA-1000, 0.5 mlCC-3202 EGM-2 MV BulletKit Medium Microvascular Endothelial Cell Growth Medium-2 BulletKit Medium w/ 5% FBSCC-3156 EBM-2 Basal Medium-2 Endothelial Cell Basal Medium-2, Serum-freeCC-4147 EGM-2 MV SingleQuots Kit Formulates EBM-2 to EGM-2 MV; hEGF, 0.5 ml; Hydrocortisone, 0.2 ml; FBS, 25 ml; VEGF, 0.5 ml; hFGF-B, 2ml; R3-IGF-1, 0.5 ml; Ascorbic Acid, 0.5 ml; GA-1000, 0.5 ml (No BBE)CC-3024 EGM Complete Medium Endothelial Cell Growth Medium w/ 2% FBSCC-3124 EGM BulletKit Medium Endothelial Cell Growth Medium BulletKit Medium w/ 2% FBSCC-3121 EBM Basal Medium Endothelial Cell Basal Medium, Serum-freeCC-3129 EBM-PRF EBM w/o Phenol RedCC-4133 EGM SingleQuots Kit Formulates EBM to EGM; BBE, 2 ml; hEGF, 0.5 ml; Hydrocortisone, 0.5 ml; FBS, 10 ml; GA-1000, 0.5 mlCC-3162 EGM-2 BulletKit Medium Endothelial Cell Growth Medium-2 BulletKit Medium w/ 2% FBSCC-3156 EBM-2 Basal Medium-2 Endothelial Cell Basal Medium-2, Serum-freeCC-4176 EGM-2 SingleQuots Kit Formulates EBM-2 to EGM-2; hEGF, 0.5 ml; Hydrocortisone, 0.2 ml; FBS, 10 ml; VEGF, 0.5 ml; hFGF-B, 2 ml;R3-IGF-1, 0.5 ml; Ascorbic Acid, 0.5 ml; GA-1000, 0.5 ml, Heparin, 0.5 ml (No BBE)00190860 EBM-2 Basal Medium (1L) Endothelial Cell Basal Medium-2 (1L)396North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Fibroblast Cell Media OptionsFibroblast media has been optimized for the proliferation offibroblasts. Each component of the basal medium and eachgrowth supplement is carefully titered for optimal growthby our R&D team. We currently offer three media choices forthe growth of fibroblasts, allowing for desired performanceand formulation flexibility. When selecting a medium to use,refer to specific media recommendations or call ourScientific Support for assistance.Product InformationCat. No. Product DescriptionFGM BulletKit Medium––FGM is a defined medium system and does not containserumFGM-2 BulletKit Medium-2––Contains a vial of FBS for a final serum concentrationof 2%FGM-3 BulletKit Medium-3––Specially formualted for Cardiac Fibroblast growth.Contains a vial of FBS for a final serum concentration of10%CC-3132 FGM-2 BulletKit Medium-2 Fibroblast Growth Medium BulletKit-2, w/ 2% FBSCC-3131 FBM Basal Medium Fibroblast Basal MediumCC-4126 FGM-2 SingleQuots Kit Formulates FBM to FGM-2; hFGF-B, 0.5 ml; Insulin, 0.5 ml FBS, 10 ml; GA-1000, 0.5 mlCC-3130 FGM BulletKit Medium Fibroblast Growth Medium BulletKit, DefinedCC-3131 FBM Basal Medium Fibroblast Basal MediumCC-4134 FGM SingleQuots Kit Formulates FBM to FGM; hFGF-B, 0.5 ml; Insulin, 0.5 ml; GA-1000, 0.5 mlCC-4526 FGM-3 BulletKit Medium-3 Fibroblast Growth Media BulletKit for cardiac fibroblastCC-4525 FGM-3 SingleQuots Kit Fibroblast Growth Media Media SingleQuots Supplements and Growth Factors for cardiac fibroblasts,Insulin, 0.5ml; rhFGF-B, 0.5 ml; GA-1000, 0.5 ml; FBS, 50 mlTechnical Information / Primary Cell Culture and MediaHepatocyte MediaProduct InformationCat. No. Product DescriptionCC-3198 HCM BulletKit Medium Hepatocyte Culture Medium, Phenol red-freeCC-3199 HBM Basal Medium Hepatocyte Basal Medium, Phenol red-free, Serum-freeCC-4182 HCM SingleQuots Kit Formulates HBM to HCM; hEGF, 0.5 ml; Transferrin 0.5 ml; Hydrocortisone, 0.5 ml; BSA, 10.0 ml;Ascorbic Acid 0.5 ml; GA-1000, 0.5 ml, Insulin, 0.5 mlCC-3197 HMM Medium HMM, Hepatocyte Maintenance Medium, Phenol red-free, Serum-freeCC-4192 HMM SingleQuots Kit HMM SingleQuots, required supplements for use <strong>with</strong> HMM; to provide optimal maintenance of cells,Insulin; 0.5 ml Dexamethasone, 0.5 ml; GA-1000, 0.5 ml9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com397
Keratinocyte Cell Media OptionsTechnical Information / Primary Cell Culture and Media9Keratinocyte media has been optimized for the proliferationof keratinocytes. Each component of the basal medium andeach growth supplement is carefully titered for optimalgrowth by our R&D team. We currently offer three mediachoices for the growth of keratinocytes, allowing for desiredperformance and formulation flexibility. When selecting amedium to use, refer to specific media recommendations inthe cell systems sections or call Lonza Scientific Support forassistance.KGM-Gold BulletKit Medium––Optimized for Clonetics NHEK – Normal HumanEpidermal Keratinocytes in a serum-free environment––KGM-Gold BulletKit Medium includes basal medium<strong>with</strong> all supplements and growth factors in separate,frozen aliquots––KGM-Gold can be used to grow all Clonetics NHEKNormal Human Epidermal Keratinocytes and provides anutrient rich medium that provides normal physiologicalgrowth characteristics and population doubling timesProduct InformationCat. No. Product DescriptionKGM-2 BulletKit Medium––Supports proliferation of primary human keratinocytesin culture––KGM-2 BulletKit Medium includes the basal mediumand supplements needed for growthKGM-CD BulletKit Medium––Supports isolation and proliferation of primary humankeratinocytes in culture––Chemically defined – No serum and no animal or plantextracts––Minimized variable experimental results due tounknown effects of animal or plant-derived components––Obtain cleaner and more accurate results quickly00192060 KGM-Gold BulletKit Medium Keratinocyte Growth Media BulletKit Medium00192151 KGM-Gold Basal Medium Keratinocyte Basal Medium, 500ml00192152 KGM-Gold SingleQuots Kit Keratinocyte Growth Medium SingleQuots; Hydrocortisone, 0.5ml, Transferrin, 0.5ml, Epinephrine,0.25ml, GA-1000, 0.5ml, BPE, 2.0ml, hEGF, 0.5ml, Insulin, 0.5ml00195769 KGM-Gold BulletKit Medium w/o Ca ++ Keratinocyte Growth Medium Calcium Free and Phenol Red Free BulletKit00195130 KGM-Gold Basal Medium w/o Ca ++ Keratinocyte Basal Medium – Calcium Free and Phenol Red FreeCC-3107 KGM-2 BulletKit Medium-2 Keratinocyte Growth Medium-2 BulletKit, Serum-freeCC-3103 KGM-2 Basal Medium-2 Keratinocyte Basal Medium-2CC-3108 KGM-2 w/o Ca ++ BulletKit Medium-2 Keratinocyte Growth Medium-2 BulletKit, Calcium-Free, Serum-freeCC-3158 KBM-2 w/o Ca ++ Basal Medium Keratinocyte Basal Medium-2, Calcium-Free, Serum-freeCC-4152 KGM-2 SingleQuots Kit Formulates KBM-2 to KGM-2; BPE, 2 ml; hEGF, 0.5 ml; Insulin, 0.5 ml; Hydrocortisone, 0.5 ml;Epinephrine, 0.5 ml; Transferrin, 0.5 ml; GA-1000, 0.5 mlCC-4455 KGM-CD BulletKit Medium Keratinocyte Growth Media – Serum-free, Non-animal origin componentsCC-3255 KBM-CD Basal Medium Keratinocyte Basal Media – Chemically DefinedCC-4456 KGM-CD SingleQuots Kit Formulates KBM-CD to KGM-CD; Recombinant human insulin 1 ml; growth supplement, 5 ml398North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Mammary Epithelial Cell Media, Serum-freeProduct InformationCat. No. Product DescriptionCC-3051 MEGM Complete Medium Mammary Epithelial Cell Growth Medium, Serum-free, complete mediumCC-3150 MEGM BulletKit Medium Mammary Epithelial Cell Growth Medium BulletKit, Serum-freeCC-3151 MEBM Basal Medium Mammary Epithelial Cell Basal Medium, Serum-freeCC-3153 MEBM-PF free Mammary Epithelial Cell Basal Medium Phenol Red Free, Serum-freeCC-3152 MEBM-Bicarb free Mammary Epithelial Cell Basal Medium Sodium Bicarbonate Free, Serum-freeCC-4136 MEGM SingleQuots Kit Formulates MEBM to MEGM; BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Insulin, 0.5 ml;GA-1000, 0.5 mlMelanocyte Cell Medium, Serum-freeClonetics Melanocyte Cell Medium has been optimized forthe growth and proliferation of normal human primarymelanocytes in culture. This medium system has beenshown to deliver superior results as compared to otherexisting commercial media systems. Melanocytes in culturemaintain >90% functionality based on the conversion ofL-dopa to dopa-melanin. The melanocyte media system alsoallows for normal morphology and proliferative capacity afterrecovery from cryopreservation and throughout serialpassaging.Product InformationCat. No. Product DescriptionMGM-4 BulletKit Medium-4––Melanocyte Growth Media has been qualified and testedtogether <strong>with</strong> NHEM to provide optimum performance––The media system is offered as a BulletKit Medium(basal medium and separately packaged growthfactors) to allow for flexibility <strong>with</strong> your research project––Adult melanocytes also require the addition of Et-3(CC-4510), sold separatelyCC-3249 MGM BulletKit Medium Melanocyte Growth MediumCC-3250 MBM-4 Basal Medium-4 Melanocyte Basal Medium, Serum-freeCC-4435 MGM-4 SingleQuots Kit Formulates MBM-4 to MGM-4; CaCl 2, 1.0 ml; hFGF-B, 1.0 ml; PMA, 0.5 ml; rhInsulin, 1.0 ml;Hydrocortisone, 0.5 ml; BPE, 2.0 ml; FBS, 2.5 ml; Gentamicin/Amphotericin B, 0.5 mlTechnical Information / Primary Cell Culture and MediaNeural Cell Medium, Low SerumProduct InformationCat. No. Product DescriptionCC-3186 AGM BulletKit Medium Astrocyte Growth Medium BulletKit, w/3.0% FBSCC-3187 ABM Basal Medium Astrocyte Basal Medium, Serum-free, <strong>with</strong>out L-glutamineCC-4123 AGM SingleQuots Kit Formulates ABM, to AGM; Insulin, 1.25 ml; rhEGF, 0.5 ml; FBS, 15.0 ml; Ascorbic Acid, 0.5 ml;L-glutamine, 5.0 ml; GA-1000, 0.5 ml9Sertoli Cell MediumProduct InformationCat. No. Product Description00191053 SeGM BulletKit Medium Sertoli Cell Growth Medium BulletKit Medium00191051 SeBM Sertoli Cell Basal Medium Sertoli Cell Basal Medium00191052 SeGM SingleQuots Kit Formulates SeBM to SeGM; FBS, 25 ml and GA-1000, 0.5 mlEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com399
Prostate Epithelial Cell Medium, Serum-freeTechnical Information / Primary Cell Culture and MediaProduct InformationCat. No. Product DescriptionCC-3166 PrEGM BulletKit Medium Prostate Epithelial Cell Growth Medium BulletKit, Serum-freeCC-3165 PrEBM Basal Medium Prostate Epithelial Cell Basal Medium, Serum-freeCC-4177 PrEGM SingleQuots Kit Formulates PrEBM to PrEGM; BPE, 2.0 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml;Transferrin, 0.5 ml; Insulin, 0.5 ml; Retinoic Acid, 0.5 ml; Triiodothyronine, 0.5 ml; GA-1000, 0.5 mlCC-4515 rCGM Rat Cardiac Myocyte GrowthMedium BulletKitIncludes basal medium and SingleQuots KitRenal Cell Media, Low SerumProduct InformationCat. No. Product DescriptionCC-3190 REGM BulletKit Medium Renal Epithelial Cell Growth Medium BulletKit, w/ 0.5% FBSCC-3191 REBM Basal Medium Renal Epithelial Cell Basal Medium, Serum-freeCC-4127 REGM SingleQuots Kit Formulates REBM to REGM; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; FBS, 2.5 ml; Epinephrine, 0.5 ml;Triiodothyronine, 0.5 ml; Transferrin, 0.5 ml; Insulin, 0.5 ml; GA-1000, 0.5 mlCC-3146 MsGM BulletKit Medium Mesangial Cell Growth Medium BulletKit, w/ 5% FBSCC-3147 MsBM Basal Medium Mesangial Cell Basal Medium, Serum-freeCC-4146 MsGM SingleQuots Kit Formulates MsBM to MsGM; FBS, 25 ml; GA-1000, 0.5 mlRetinal Pigment Epithelial Cell MediaProduct InformationCat. No. Product Description00195409 RtEGM BulletKit Medium Retinal Pigment Epithelial Cell Medium BulletKit Medium00195406 RtEBM Basal Medium Retinal Pigment Epithelial Cell Basal Medium00195407 RtEGM SingleQuots Kit Formulates RtEBM to RtEGM; L-glutamine, 4 ml; FBS, 4 ml; bFGF, 1 ml; GA-1000, 0.5 mlPrimary Neuron Growth Medium, Serum-free9Product InformationCat. No. Product DescriptionCC-4461 PNGM BulletKit Medium Primary Neuron Growth Medium BulletKit MediumCC-3256 PNBM Basal Medium Primary Neuron Basal MediumCC-4462 PNGM SingleQuots Kit Formulates PNBM to PNGM; NSF-1, 4 ml; L-glutamine, 2 ml; GA-1000, 0.2 mlCC-4512 PNGM-A BulletKit Medium Primary Neuron Growth Media-Adult (contains CC-3256, CC-4462, and CC-4511)CC-4511 PNGM-A SingleQuots Kit Formulates PNGM to PNGM-Adult; OA, 0.5 ml; PA, 1.5 ml400North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Human Mesenchymal Stem Cell MediaProduct InformationCat. No. Product DescriptionPT-3001 MSCGM BulletKit Medium Mesenchymal Stem Cell Growth Medium BulletKit MediumPT-3238 MSCGM Basal Medium Mesenchymal Stem Cell Basal MediumPT-4105 MSCGM SingleQuots Kit Formulates MSCBM to MSCGM Growth Medium; MCGS, 50 ml; L-glutamine, 10 ml; GA-1000, 0.5 mlPT-3002 hMSC Differentiation Kit– Osteogenic Mesenchymal Stem Cell Differentiation Kit – OsteogenicPT-3924 hMSCBM – Osteogenic Basal Medium Mesenchymal Stem Cell Basal Medium – OsteogenicPT-4120 hMSC SingleQuots Kit – Osteogenic Formulates Osteogenic Basal Medium to Osteogenic Differentiation Medium; Dexamethasone, 1 ml;ß-Glycerophosphate, 2 ml; Ascorbate, 1 ml; Penicillin/Streptomycin, 2 ml; MCGS, 20 ml;L-glutamine, 4 mlPT-3003 hMSC Differentiation Kit – Chondrogenic Mesenchymal Stem Cell Differentiation Kit – ChondrogenicPT-3925 hMSCBM – Chondrogenic Basal Medium Mesenchymal Stem Cell Basal Medium – ChondrogenicPT-4121 hMSC SingleQuots Kit – Chondrogenic Formulates Chondrogenic Basal Medium to Chondrogenic Differentiation Medium; ITS + , 2 ml; SodiumPyruvate, 2 ml; Proline, 2 ml; Dexamethasone, 1 ml; Ascorbate, 2 ml; GA-1000, 0.2 ml; L-glutamine, 4 mlPT-4124 TGF-ß3 Required component sold separatelyPT-3004 hMSC Differentiation Kit – Adipogenic Mesenchymal Stem Cell Differentiation Kit – AdipogenicPT-3102A hMSC Adipogenic Maintenance Medium Mesenchymal Stem Cell Maintenance Medium – AdipogenicPT-3102B hMSC Adipogenic Induction Medium Mesenchymal Stem Cell Induction Medium – AdipogenicPT-4122 hMSC Maintenance SingleQuots Kit –AdipogenicPT-4135 hMSC Induction SingleQuots Kit –AdipogenicRat Mesenchymal Stem Cell MediaProduct InformationCat. No. Product DescriptionFormulates Adipogenic MM to Adipogenic Differentiation Medium; rhInsulin, 2 ml; GA-1000, 0.2 ml;MCGS, 20 ml; L-glutamine, 4 mlFormulates Adipogenic M to Adipogenic Differentiation Medium; Indomethacin, 0.4 ml; IBMX, 0.2 ml;rhInsulin, 2 ml; Dexamethasone, 1 ml; GA-1000, 0.2 ml; MCGS, 20 ml; L-glutamine, 4 ml00192853 Rat MSC Growth Medium BulletKit Medium Rat Mesenchymal Stem Cell Growth Medium BulletKit MediumPT-3238 MSCGM Basal Medium Mesenchymal Stem Cell Basal MediumPT-4105 R-MSCGM SingleQuots Kit Formulates MSCBM to Rat MSC Growth Medium; FBS, 50 ml; rhInsulin, 2 ml; L-glutamine, 10 ml;GA-1000, 0.5 ml00192855 rMSC Differentiation Kit – Adipogenic Rat Mesenchymal Stem Cell Differentiation Kit – AdipogenicPT-3102A MSC Adipogenic Maintenance Medium Mesenchymal Stem Cell Maintenance Medium – AdipogenicPT-3102B MSC Adipogenic Induction Medium Mesenchymal Stem Cell Induction Medium – Adipogenic00192828 rMSC Maintenance SingleQuots Kit –Adipogenic00192827 hMSC Induction SingleQuots Kit –AdipogenicFormulates Adipogenic MM to Adipogenic Differentiation Medium; rhInsulin, 2 ml; GA-1000, 0.2 ml;FBS, 20 ml; L-glutamine, 4 mlFormulates Adipogenic M to Adipogenic Differentiation Medium; Indomethacin, 0.4 ml; IBMX, 0.2 ml;rhInsulin, 2 ml; Dexamethasone, 1 ml; GA-1000, 0.2 ml; FBS, 20 ml; L-glutamine, 4 ml00192854 rMSC Differentiation Kit– Osteogenic Rat Mesenchymal Stem Cell Differentiation Kit – OsteogenicPT-3924 hMSCBM – Osteogenic Basal Medium Mesenchymal Stem Cell Basal Medium – Osteogenic00192829 rMSC SingleQuots Kit – Osteogenic Formulates Osteogenic Basal Medium to Osteogenic Differentiation Medium; Dexamethasone, 1 ml;ß-Glycerophosphate, 2 ml; Ascorbate, 1 ml; Penicillin /Streptomycin, 2 ml; FBS, 20 ml;L-glutamine, 4 mlTechnical Information / Primary Cell Culture and Media9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com401
Human Adipose-Derived Stem CellsTechnical Information / Primary Cell Culture and MediaProduct InformationCat. No. Product DescriptionPT-4505 ADSC-GM BulletKit Medium Adipose-Dervived Stem Cell Growth Medium BulletKit MediumPT-3273 ADSC Basal Medium Adipose-Derived Stem Cell Basal MediumPT-4503 ADSC-GM SingleQuots Kit Formulates ADSCBM to ADSC Growth Medium; FBS, 50 ml; L-glutamine, 5 ml; GA-1000, 0.5 mlPreadipocyte Growth MediumProduct InformationCat. No. Product DescriptionPT-8002 PGM-2 BulletKit Medium Preadipocyte Growth Medium-2 BulletKit MediumPT-8202 PBM-2 Basal Medium-2 Preadipocyte Basal Medium-2PT-9502 PGM-2 SingleQuots Kit Formulates PBM-2 to PGM-2; FBS (10%), 50 ml; L-Glutamine, 5 ml; GA-1000, 0.2 ml, 5 ml;rhInsulin, 2 ml; Dexamethasone, 0.2 ml; Indomethacin, 0.4 ml; IBMX, 0.2 mlOsteoclast Growth MediumProduct InformationCat. No. Product DescriptionPT-8001 Osteoclast Growth Medium BulletKit Osteoclast Growth Medium BulletKit MediumMediumPT-8201 OPBM Basal Medium Osteoclast Basal MediumPT-9501Osteoclast Growth MediumSingleQuots KitFormulates OPBM to Osteoclast Growth Medium; FBS (10%), 10 ml; L-glutamine, 1 ml; Penicillin/Streptomycin, 1 ml; M-CSF, 0.1 ml; Soluble RANK Ligand, 2 μgNeural Progenitor Growth Medium9Product InformationCat. No. Product DescriptionCC-3209 NPMM BulletKit Medium Neural Progenitor Maintenance Medium BulletKit MediumCC-3210 NPBM Basal Medium Neural Progenitor Basal MediumCC-4242 NPDM SingleQuots Kit Formulates NPBM to NPMM (for differentiation) NSF-1, 4 ml; GA-1000, 0.4 mlCC-4241 NPMM SingleQuots Kit Formulates NPBM to NPMM (for maintenance) rhEGF, 0.4 ml; rhFGF, 0.4 mlCC-3229 NPDM BulletKit Medium Neural Progenitor Differentiation Medium BulletKit MediumCC-3210 NPBM Basal Medium Neural Progenitor Basal MediumCC-4242 NPDM SingleQuots Kit Formulates NPBM to NPDM (for differentiation); NSF-1, 4 ml; GA-1000, 0.4 ml402North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Skeletal Cell MediumProduct InformationCat. No. Product DescriptionCC-3207 OGM BulletKit Medium Osteoblast Growth Medium BulletKit, w/ 10% FBSCC-3208 OBM Basal Medium Osteoblast Cell Basal Medium, Serum-freeCC-4193 OGM SingleQuots Kit Formulates OBM to OGM; FBS, 50 ml; Ascorbi Acid, 0.5 ml; GA-1000, 0.5 mlCC-4194 OGM Differentiation SingleQuots Kit Induces osteoblast differentiation and bone mineralization; Hydrocortisone-21-Hemisuccinate, 0.5 ml;ß-Glycerophosphate, 5.0 mlCC-3216 CGM BulletKit Medium Chondrocyte Growth Medium BulletKit MediumCC-3217 CBM Basal Medium Chondrocyte Basal MediumCC-4409 CGM SingleQuots Kit Formulates CBM to CGM; R3-IGF-1, 1.0 ml; bFGF, 2.5 ml; Insulin, 1.0 ml; Transferrin, 0.5 ml; FBS, 25 mlCC-3225 CDM BulletKit Medium Chondrocyte Differentiation Medium BulletKit, Serum-freeCC-3226 CDM Basal Medium Chondrocyte Differentiation Basal MediumCC-4408 CDM SingleQuots Kit Formulates CDM Basal Medium to CDM Differentiation Medium; TGF-β, 1.25 ml; R3-IGF-1, 0.5 ml;Insulin, 0.5 ml; Transferrin, 0.5 ml; FBS, 12.5 ml; GA-1000, 0.25 mlSkeletal Muscle Cell MediumProduct InformationCat. No. Product DescriptionCC-3160 SkGM BulletKit Medium Skeletal Muscle Growth Medium BulletKit, Serum-freeCC-3161 SkBM Basal Medium Skeletal Muscle Basal Medium, Serum-freeCC-4139 SkGM SingleQuots Kit Formulates SKBM to SKGM; hEGF, 0.5 ml; Insulin, 5 ml; BSA, 5 ml; Fetuin, 5 ml ; Dexamethasone, 0.5ml; GA-1000, 0.5 mlCC-3245 SkGM-2 BulletKit Medium-2 Skeletal Muscle Myoblast Growth Medium-2 BulletKit, <strong>with</strong> 10% FBSCC-3246 SkBM-2 Basal Medium-2 Skeletal Muscle Myoblast Basal Medium-2, Serum-free; no L-glutamineCC-3244 SkGM-2 SingleQuots Kit Formulates SkBM-2 to SkGM-2; FBS, 50.0 ml; GA-1000, 0.5 ml; rhEGF, 0.5 ml; Dexamethasone, 0.5 ml;L-glutamine, 10.0 mlTechnical Information / Primary Cell Culture and MediaSmooth Muscle Cell MediumProduct InformationCat. No. Product DescriptionCC-3182 SmGM-2 BulletKit Medium-2 Smooth Muscle Growth Medium-2 BulletKit Medium w/ 5% FBSCC-3181 SmBM Basal Medium Smooth Muscle Basal Medium, Serum-freeCC-4149 SmGM-2 SingleQuots Kit Formulates SmBM to SmGM-2; hEGF, 0.5 ml; Insulin, 0.5 ml; hFGF- B, 1 ml; FBS, 25 ml;GA-1000, 0.5 ml9Stromal Cell Medium, Low SerumProduct InformationCat. No. Product DescriptionCC-3205 SCGM BulletKit Medium Stromal Cell Growth Medium BulletKit, w/ 5% FBSCC-3204 SCBM Basal Medium Stromal Basal Medium, w/o phenol red, Serum-freeCC-4181 SCGM SingleQuots Kit Formulates SCBM to SCGM; hFGF-B, 0.5 ml; Insulin 0.5 ml; FBS, 25 ml; GA-1000, 0.5 mlEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com403
Primary Cell MethodsTechnical Information / Primary Cell MethodsProcedure for Thawing Mononuclear Cells andHematpoeitic Progenitor Cells1. Warm medium containing 10% FBS or 1% BSA. Formononuclear cells and hematopoietic progenitors,DNase I (20 U/ml) should also be added.*2. Quickly thaw the vial of frozen cells in a 37°C water bath.Wipe the outside of the vial <strong>with</strong> 70% ethanol.3. Aseptically transfer a maximum of 2 ml of cellsuspension to a 50 ml conical tube. For 1 million cells orless, use a 15 ml conical tube.4. Rinse the vial <strong>with</strong> 1 ml of medium. Add the rinsedropwise to the cells while gently swirling the tube(≈1 minute).5. Slowly add enough medium dropwise to the cells untilthe total volume is 5 ml, while gently swirling after eachaddition of several drops of medium (≈3 minutes).6. Slowly bring the volume up to fill the tube by adding1 ml to 2 ml volumes of medium dropwise, while gentlyswirling after each addition of medium (≈5–10minutes).7. Centrifuge the cell suspension at 200 × g at roomtemperature for 15 minutes.8. Carefully remove by pipette (and save in a secondtube) most of the wash, leaving a few mls behind so thecell pellet is not disturbed. Gently resuspend the cellpellet in the remaining medium. If you are using a 50 mltube, transfer the cells to a 15 ml conical tube and rinsethe 50 ml tube <strong>with</strong> 5 ml of medium. Slowly add the 5 mlwash medium to the cell suspension <strong>with</strong> gentleswirling.9. Slowly bring the volume up to fill the tube by adding1 ml to 2 ml volumes of medium while gently swirlingafter each addition of medium.10. Centrifuge the cell suspension at 200 × g at roomtemperature for 15 minutes.11. Carefully remove by pipette all but 2 ml of the wash.Gently resuspend the cell pellet in the remaining 2 ml ofmedium and count. If cell count is lower than expected,centrifuge the wash saved in step 8 at a higher speed,count and combine if necessary.12. Rest the cells for 1 hour at 37°C and 5% CO 2. Count thecells a second time. The cells are ready to be put inculture.*For the addition of DNase, prepare 20 ml of medium containing 10%FBS and 20 U/ml of DNase I (Sigma D 4513). Proceed as above, usingthe DNase-containing medium to dilute the cells. Centrifuge the cellsand continue <strong>with</strong> step 8.10404North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Culture Set-up – Adherent Cell TypesThese Instructions do not Apply to All Cell Types.Please go to:www.lonza.com/cellbioinstructionsfor detailed, cell specific instructions.1. Calculate the number of vessels to be set up. Refer toyour Certificate of Analysis for the exact number of cellsin your cryovial. Refer to table on page 377 “Growth Areaof Common Plasticware”, for help in adjusting thiscalculation.Use the following calculations to determine the number of vessels tobe set-up for the recommended seeding densities of 2,500 cells/cm 2 ,3,500 cells/cm 2 , or 5,000 cells/cm 2No. of cells available × Percent viability =Max. no. of cm 2 thatcan be platedRecommended seeding densityMax. no. of cm 2 that can be platedEffective growth area of flask=Max. no. of flasks thatcan be set upExample: A cryovial of HMVEC-L <strong>with</strong> 520,000 cells and 80% viability520,000 × 0.80= 83 cm 2 , to be set up5,000If you use a T-25 <strong>with</strong> an effective growth area of 25 cm 283 cm 2 =25 cm 23 flasks (rounded down to the nearestwhole number of flasks)The advantage of setting up this number of T-25 flasksfrom the initial cryovial, as opposed to larger flasks, isthat it reduces the risk of losing large numbers of cells.That is, if you experience difficulty trypsinizing the firstT-25 flask, there are other remaining T-25 flasks to use.2. Label each flask <strong>with</strong> the passage number, cell type, lotnumber, and date.3. In a sterile field, carefully open the supplemented bottleof growth medium and aseptically transfer the mediumto new culture vessels by adding 1 ml growth mediumfor every 5 cm 2 surface area of the flask.Example: 5 ml growth medium for a 25 cm 2 flask.4. Tighten vented caps on vessels. If vented caps are notbeing used, twist caps until tight, then loosen about 1/2turn. Allow the culture vessels to warm and equilibratein a 37°C, 5% CO 2, humidified incubator for at least 30minutes.Technical Information / Primary Cell MethodsThawing Cells – Adherent Cell TypesAseptically Add the Recommended Amount of Mediumto the Flask and Equilibrate for 30 Minutes in a 5% CO 2,37°C Incubator1. Have a micropipette ready prior to thawing.2. Remove the cryovial of cells from storage. Wipe cryovial<strong>with</strong> ethanol or isopropanol before opening. In a sterilefield, briefly twist the cap a quarter turn to relieve theinternal pressure, then retighten; do not open thecryovial completely.3. Holding the cryovial, dip the bottom 3/4 of the cryovialin a 37°C water bath and swirl gently for 1–2 minutesuntil contents are thawed. Watch the cryovial closely;when the last sliver of ice melts remove it; DO NOTsubmerge it completely. Thawing the cells for longerthan 2 minutes may result in less than optimal results.4. Remove the cryovial immediately, wipe it dry, andtransfer to a sterile field where the equilibrated flasksshould be waiting, ready to seed. Rinse the cryovial <strong>with</strong>70% alcohol, then wipe to remove excess.5. Note the color of the thawed cryovial. Ideally, the color ofthe thawed cryovial should be pink. If the color is notpink, seed the cells, note the color and mention this factto Scientific Support if seeding is not successful.NOTES:––If more than one cryovial is to be thawed, thaw one cryovial at a timeand keep other cryovials in liquid nitrogen until ready for use––Cryopreserved cells are very delicate; thaw and return them to cultureas quickly as possible <strong>with</strong> minimal handling––Wear eye protection when handling frozen cells; rapid temperaturechanges may cause splattering of liquid nitrogen––Centrifugation should not be performed to remove cells from thecryoprotectant cocktail; this action is more damaging than the effectsof DMSO residue in the culture––It is not recommended to thaw frozen cells directly onto glass slides,chamber slides, gridded plates or multiwell plate configurations (6, 12,24, 96…); optimal performance is achieved when initial seeding outof cryopreservation is performed into T-25 flasks; for further instructionsfollow the directions in the set-up section in the cell culture instructionsprovided or contact Scientific Support for cell-specific protocols10Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com405
Seeding – Adherent Cell TypesTechnical Information / Primary Cell MethodsRemove the Cap, Being Careful Not to Touch the InteriorThreads <strong>with</strong> Your Fingers1. Using a 1,000 µl micropipette set to 800 µl, put the tipinto the cryovial and resuspend the cells <strong>with</strong> a gentle,slow and steady up and down pipetting motion no morethan five times. DO NOT resuspend quickly and keep thetip near the bottom to avoid making bubbles.2. Dispense an equal amount of cells into the flasks set upearlier (as determined by the recommended seedingdensity and number of cells/vial (see page 391)). Iffour T-25 flasks were prepared, set micropipette to 250µl and dispense. If eight T-25 flasks were prepared, setmicropipette to 125 µl and dispense.NOTE: Do not dispense the entire contents of the cryovial into one T-25flask!3. Replace the cap or cover and gently rock the vessels toevenly distribute the cells. Loosen caps if necessary topermit gas exchange.4. Return the culture vessels to a 37°C incubator <strong>with</strong>5% CO 2. Lay them flat on the shelf, providing the largestsurface for cells to attach. The cells will anchor to thebottom surface of the flask.After SeedingCells are not tolerant to rapid temperature fluctuations ornutrient-deficient medium. Feeding them <strong>with</strong> fresh growthmedium that has been warmed will avert potential problems.(Remember to warm only the amount needed.) Check andfeed the cells on the schedule below, even on weekends andholidays.5. Change the growth medium the day after seeding (toremove residual DMSO and unattached cells), thenevery other day thereafter while examining them daily.NOTE: A change of medium requires removal of the medium byaspirating <strong>with</strong> a sterile pipette on the opposite side of the flask fromwhere the cells are attached. Then warm, fresh medium is added downthe same side.6. Successfully recovered cultures will exhibit thefollowing:6.1 Cells <strong>with</strong> clear non-granular cytoplasm.6.2 Numerous mitotic figures after day 2.7. Feed the cells a larger volume of medium as theybecome more confluent. Use this table as a guideline:If cells are:Then feed them:Under 25% confluent 1 ml per 5 cm 2From 25–45% confluent 1.5 ml per 5 cm 2Exceeding 45% confluence 2 ml per 5 cm 28. Continue feeding the cells until 60–90% confluence. Ifspecific cell types are allowed to become over-confluent(i.e., epithelial cells) and stay at confluence for morethan 2 days, they can suffer irreversible contactinhibition and may detach from the flask and/or bedifficult to trypsinize.Proliferating Cells – Adherent Cell Types101. Examine the culture microscopically for any signsof distress during shipment (i.e., detachment,rounding-up, or atypical morphology). Check therelative cell density and estimate % confluency. Theculture should be 30–100% confluent upon receipt.Some cellular detachment is normal. Please callScientific Support immediately if cells look severelydistressed.2. Decontaminate the external surface of the cell cultureflask or multiwell dish by wiping <strong>with</strong> 70% ethanol orisopropanol.3. Incubate the sealed flask or multiwell dish at 37°C,5% CO 2, for 3–4 hours to equilibrate temperature.4. Warm an appropriate amount of growth medium to 37°Cin a sterile container. Warming the entire bottle canshorten the life of the medium. Never warm mediumunder hot running water or any other uncontrolledtemperature source. Never microwave.5. In a sterile field, carefully open the cell culture flask ormultiwell dish, remove the medium and replace it <strong>with</strong>the warmed, fresh medium. Aseptically remove anymedium inside the neck or cap area because it canfacilitate microbial contamination.6. If you are using a flask <strong>with</strong> a non-vented cap, loosenthe cap and return the flask to the 37°C humidifiedincubator <strong>with</strong> 5% CO 2for at least 24 hours.406North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Subculturing – Adherent Cell TypesStorage Information for Subculture Reagents1. Subculture reagents are sterile-filtered and then storedat -20°C until shipped from our Distribution Centers.2. Subculture reagents may thaw during transport. Theymay be refrozen once.3. Subculture reagents can be stored at -20°C untilexpiration date, after thawing once and refreezing.4. To keep Trypsin/EDTA fresh and active after thawing,you may aliquot it into sterile centrifuge tubes andrefreeze at -20°C. Trypsin/EDTA may be stored frozenuntil the expiration date.5. We recommend that HEPES-BSS and the TrypsinNeutralization Solution once stored at 4°C be used<strong>with</strong>in one month.PreparationThe following instructions are for a 25 cm 2 flask (T-25).Adjust all volumes accordingly for other size flasks.Preparation for subculturing the first flask:6. Subculture the cells when they are 60–90% confluentand contain many mitotic figures throughout the flask.7. For each 25 cm 2 of cells to be subcultured:7.1 Thaw 2 ml of Trypsin/EDTA and allow to come toroom temperature.7.2 Allow 7–10 ml of HEPES Buffered Saline Solution(HEPES-BSS) to come to room temperature.7.3 Allow 4 ml of Trypsin Neutralizing Solution (TNS)to come to room temperature.8. Remove growth medium from 4°C storage and allow tostart warming to room temperature.9. Prepare new culture vessels from 9.3 on work in asterile field:9.1 Prepare 1–3 T-75 flasks. The number of flasksneeded depends upon confluence and total yield.Larger flasks may be used to save plasticwareand time spent on subsequent subcultures.Smaller flasks reduce the risk of losing asubstantial part of your culture.9.2 As before, label each flask <strong>with</strong> the passagenumber, lot number,cell type, and date.9.3 In a sterile field, carefully open the bottle andtransfer growth medium to new culture vesselsby adding 1 ml growth medium for every 5 cm 2surface area of the flask.Example: 15 ml growth medium for a 75 cm 2 flask9.4 If not using vented caps, loosen caps of flasks.Place the new culture vessels into a 37°Chumidified incubator <strong>with</strong> 5% CO 2and equilibratethe flasks for at least 30 minutes. Subculture oneflask at a time. All flasks following the first flaskwill be subcultured following an optimization ofthis protocol (explained later in this procedure),based on calculated cell count, cell viability, andseeding density.NOTE: Use only Clonetics Trypsin/EDTA. The concentration of Trypsin/EDTA from other suppliers may be 10X our concentration, which willdetrimentally effect Clonetics Cells.10. Aspirate the medium from one culture vessel.11. Rinse the cells <strong>with</strong> 5 ml of room temperature HEPESBuffered Saline Solution (HEPES-BSS). DO NOT forgetthis step. The medium contains complex proteins thatneutralize the trypsin.12. Aspirate the HEPES-BSS from the flask.13. Cover the cells <strong>with</strong> 2 ml of Trypsin/EDTA solution.14. Tighten the cap and begin monitoring the flask underthe microscope.15. Continue to examine the cell layer microscopically.15.1 Allow the trypsinization to continue untilapproximately 90% of the cells are rounded up.NOTE: Rounded up cells are spherical, have smooth edges and arerefractile or shiny. If the cells still have protruding nubs which are stillattached to the flask, they need more time to trypsinize. This entireprocess takes about 2–6 minutes, depending on cell type.15.2 At this point, rap the flask against the palm of yourhand to release the majority of cells from theculture surface. If only a few cells detach, youmay not have let them trypsinize long enough.Wait 30 seconds and rap again. If cells still do notdetach, wait and rap every 30 seconds thereafter.NOTE: Do not try to get all cells to detach by rapping them severely. Thisaction may damage the cells.16. After cells are released, neutralize the trypsin in theflask <strong>with</strong> 4 ml of room temperature Trypsin NeutralizingSolution. If the majority of cells do not detach <strong>with</strong>inseven minutes, the trypsin is either not warm enoughor not active enough to release the cells. Harvest theculture vessel as described above, and eitherre-trypsinize <strong>with</strong> fresh, warm Trypsin/EDTA Solution orrinse <strong>with</strong> Trypsin Neutralizing Solution and then addfresh, warm medium to the culture vessel and return toan incubator until fresh trypsinization reagents areavailable.Technical Information / Primary Cell Methods10Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com407
Subculturing – Adherent Cell TypesContinuedTechnical Information / Primary Cell Methods17. Quickly transfer the detached cells to a sterile15 ml centrifuge tube.18. Rinse the flask <strong>with</strong> a final 2 ml of HEPES-BSS to collectresidual cells, and add this rinse to the centrifuge tube.19. Examine the harvested flask under the microscope tomake sure the harvest was successful by looking atthe number of cells left behind. This should be lessthan 5%.20. Centrifuge the harvested cells at 220 × g for5 minutes to pellet the cells.20.1 Aspirate most of the supernatant, except for 100µl to 200 µl.20.2 Flick the centrifuge tube <strong>with</strong> your finger to loosenthe pellet.21. Dilute the cells in 4 ml to 5 ml of growth medium andnote the total volume of the diluted cell suspension.NOTE: To obtain the best results from your cells, assess cell yieldand viability <strong>with</strong> Trypan Blue.22. Count the cells <strong>with</strong> a hemacytometer or cell counterand calculate the total number of cells. (See instructionsabove.) Make a note of your cell yield for later use.NOTE: The cell suspension should contain between 250,000 to1,000,000 cell/ml for greatest accuracy.23. If necessary, dilute the suspension <strong>with</strong> HEPES BufferedSaline Solution (HEPES-BSS) to achieve the desired“cells/ml” and re-count the cells.24. Assess cell viability using Trypan Blue.25. Use the following equation to determine the totalnumber of viable cells.Total cell count × percent viability = Total # of viable cellsExample: 1,000,000 cells × 60% = 600,000 viable cells26. Determine the total number of flasks to inoculate byusing the equation below. The number of flasks neededdepends upon cell yield and seeding density. Largerflasks may be used to save plasticware and time spenton subsequent subcultures. Smaller flasks reduce therisk of losing a substantial part of your culture ifcontamination occurs.The recommended seeding density could be 2,500 cells/cm 2 , 3,500 cells/cm 2 , or 5,000 cells/cm 2 for flasks and 10,000 cells/cm 2for well platesTotal # of viable cellsRecommended seedingdensity × flask size600,000 viable cells75 cm 2 × 2,500 cells/cm 2==Total # of flasksto inoculate3 T-75 flasks (rounded downto nearest whole number)27. Use the following equation to calculate the volume ofcell suspension to seed into your flasks.Total volume of diluted cell suspension# of flasks as determined in step 17= Seeding volume4.3 ml of diluted cell suspension = 1.43 ml per T-753 T-75 flasksflask28. Prepare flasks by labeling each flask <strong>with</strong> the passagenumber, lot number, cell type, and date.29. Carefully open the medium bottle and transfer growthmedium to new culture vessels by adding1 ml growth medium for every 5 cm 2 surface area of theflask (1 ml/5 cm 2 ).30. Example: 15 ml growth medium for a 75 cm 2 flask.31. After mixing the diluted cells <strong>with</strong> a 5 ml pipette toensure a uniform suspension, dispense the volume ofsuspension calculated above into the preparedsubculture flasks.32. After dispensing the cells, gently rock flask to promoteeven distribution.33. If not using vented caps, loosen caps of flasks. Placethe new culture vessels into a 37°C humidified incubator<strong>with</strong> 5% CO 2.10408North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Subculturing into 96-well PlatesOverviewA culture flask of human cells is harvested by tryp-sinizationand subsequent trypsin inhibitor treatment. The cells arecentrifuged, resuspended in growth medium and counted.The desired number of cells is then added to wells of sterile96-well tissue culture plates. The plates are incubated in a37°C, 5% CO 2humidified incubator for 1–3 days to allow forcell adherence and growth. Seeding densities will varysomewhat <strong>with</strong> your experimental requirements. A densityof 10,000 cells/cm 2 for multiwell plates is ideal. See the CellCulture Instruction Sheet for the cell type you are using forspecific information.Required Materials:1. T-25 flask of proliferating normal human cells between60% and 90% confluence2. 96-well flat bottom, tissue culture plates3. 37°C humidified incubator <strong>with</strong> 5% CO 2/95% air4. Laminar flow hood or other sterile environment5. Adjustable multichannel pipette (8 or 12 channel) orrepeating pipette6. Sterile reservoir(s) for use <strong>with</strong> multichannel pipetteProcedure7. Follow the steps for subculture preparation andsubculturing. Then follow steps 2–4 below.8. Since the cells/ml calculation computed is per ml, thecell concentration must be increased by 4 times beforeseeding 96-well plates (to accommodate the 1:4dilution when adding 250 µl of suspended cells perwell). When making the cell suspension, adjust the cellconcentration <strong>with</strong> growth medium.9. Transfer the diluted cell suspension to a sterilereservoir. Using a multichannel (8 or 12 channel)pipette equipped <strong>with</strong> sterile pipette tips, add 250 µl ofthe diluted cell suspension to each well of the labeled96-well flat bottom, tissue culture plate(s).NOTE: Resuspend the cell suspension often during the seedingprocedure to ensure a uniform number and distribution of cells intoeach well by pipetting up and down a few times between every otherdispensing.10. Cover and incubate the plates for 1–3 days at 37°C/5%CO 2. (Incubation periods exceeding 3 days are generallynot recommended because of evaporation of mediumfrom the edge wells of the plate).Technical Information / Primary Cell MethodsNOTE: Before using the 96-well plate culture in a bioassay, examine thecells microscopically for the presence of mitotic figures asa confirmation that the cells have resumed active growth.10Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com409
Instructions for CryopreservationTechnical Information / Primary Cell MethodsCryopreservation may compromise cell quality andperformance. Performance of the cells cannot beguaranteed after cryopreservation. These instructions donot apply to hepatocytes, bMVEC-B, intestinal epithelialcells, melanocytes, mouse and rat neuronal cells, ratcardiac cells, and Poietics Cells.Instructions1. Sterile filter freezing medium using a cell culture rated0.2-micron filter.2. Harvest cells and spin them down.3. Resuspend cells in cold freezing solution at ≈500,000to 2,000,000 cells/ml. Work quickly. Once exposed toDMSO, cells become very fragile.4. Pipet aliquots (1 ml each) into freezing vials orampoules and seal.5. Insulate aliquots <strong>with</strong> a STYROFOAM® or propanolfreezing canister.6. Store cells at -70°C overnight.7. Within 12–24 hours, place in LN2 (-200°C) for longtermstorage. Cells will be compromised by prolongedstorage at -70°C.Cryopreservation Solutions■■for Mesenchymal Stem Cells––70% MSCBM––10% DMSO––20% human serum albumin (25% solution)■■For Skeletal Muscle Cells––70% SkGM––20% FBS––10% DMSO■■For All Other Cell Types––80% Clonetics Growth Medium of choice––10% FBS––10% DMSO10410North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Improving Cell Yield and Viability During SubcultureSeveral factors, or a combination of factors, can contributeto low cell count and low cell viability. If cell yield or viabilityis unsatisfactory, use the following information to increasethe success rate of future cultures.Low Yield (Cell Count)Condition Possible Causes SolutionsImproving Cell ViabilityIf your cell viability is low (less than 50%), determine thepossible cause(s) and solution(s) using the table below.Then subculture one more flask applying the appropriatesolution(s).Improving Cell YieldIf your cell yield is low (less than 50%), determine thecause(s) and possible solution(s) using the table below.Then subculture one or more flasks applying the appropriatesolution(s).Majority of cells did not detach 1. Inactive or cold Trypsin/EDTA 1. Use Trypsin/EDTA at room temperature2. Improper storage of Trypsin/EDTA 2. Store at -20°C until ready for use; thaw and allow it to cometo room temperature briefly before subculturing3. Exposure time to Trypsin/EDTA was too short 3. Exposure time to Trypsin/EDTA is usually 5–6 minutes4. Trypsin/EDTA has been neutralized 4 .Be sure to rinse the culture completely <strong>with</strong> HEPES-BSSbefore trypsinization5. Vessel was not rapped firmly 5. Use a moderate amount of force when rapping duringtrypsinizationLow yield, 95% of the cells detached but theyield was lowCulture was under confluent at trypsinization Be sure to trypsinize at 60–90% confluence <strong>with</strong> numerousmitotic figures throughout the flaskTechnical Information / Primary Cell MethodsLow Viability (
Custom Cell Isolation ServicesTechnical Information / Primary Cell MethodsConsult the cell culture experts and harness the power ofour extensive knowledge and experience delivering customcell solutions. Choose from an endless variety of humanand animal primary cell types, cryopreserved, plated or inculture flasks, for your convenience. Save time and moneyby avoiding the aggravation of tissue acquisition, failedisolations and low yields.Quality and ExperienceAs the cell culture experts, Lonza maintains anISO 9001:2008 certified custom cell isolation laboratorystaffed by some of the world’s finest cell culture technicians,providing a variety of cell types for your individual researchneeds. Partner <strong>with</strong> us and benefit from over 60 years ofcombined cell culture isolationexperience.■■We offer:––Expert custom cell isolation solutions made to yourspecifications––Extensive quality testing and cellular characterizationavailable––Years of isolation experience <strong>with</strong> a wide variety of celltypes––Custom formats: cryopreserved, plates or flasks––State-of-the-art cell isolation capabilities■■Delays in obtaining vital primary cell cultures can bevery costly. Discover our superior custom cell isolationcapabilities including:––Primary cell isolations from human and animal tissues––Cell expansion and testing––Donor-matched cell sets––Cryopreserved or proliferating formats––Wide array of QC and cell characterization services,including PCR––Individual customer consultation to ensure correctorder fulfillment■■Custom primary cell isolations from human, rat, mouse,porcine, bovine, monkey and many other species.Examples of successful isolations include:––Bovine adrenal gland capillary endothelial cells––Bovine embryonic kidney cells––Bovine and porcine preadipocytes––Canine umbilical vein endothelial cells––Cat iris smooth muscle cells––Guinea pig kidney cells––Human bladder epithelial cells––Human bronchial/tracheal epithelial cells –Cystic Fibrosis––Human diabetic CD34 + cells––Human microvascular retinal endothelial cells––Human small intestine disassociated cells––Murine dermal fibroblasts––Porcine mesangial cells10412North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
TransfectionCell Culture Tips for Cell Lines and Primary Cells Prior to TransfectionIntroductionIn order to help ensure that your cells are in the best possiblecondition before transfection, please take a moment toreview the suggestions below. These are in no way intendedto replace product protocols, but rather to give you somehelpful hints to facilitate the success of your experiments.Passage NumberCells of a lower passage number typically respond better totransfection and will have higher transfection efficienciesand viabilities than those of higher passage numbers. Forthe most efficient gene transfer, we recommend using cellsthat are in logarithmic growth phase and at a passagenumber less than 10–15 (from the time of thaw). This isbecause some cell lines differentiate and change theirfeatures after many passages. If you are transfectingprimary cells or cell lines that have been cryopreserved, werecommend that they be passaged at least two times toallow them to begin growing properly prior to transfection.Frozen primary blood cells should be incubated in growthmedium for a minimum of 1–2 hours before transfection.Growth ConditionsAdherent cells – For the transfection of adherent cells, thecells should be generally grown to a confluency of 70–85%.The confluency of the culture prior to transfection isimportant. If the cells are allowed to grow to a higherconfluency than recommended, or up to 100% confluency,you may get transfection results other than mentioned inthe protocols. We also suggest that cells be passaged2–4 days prior to transfection, so that they achieve therequired confluency for your experiments. For transfectionof adherent cells by an electroporation-based method likeNucleofection (see page 148), cells had to be releasedfrom the culture vessel and transferred into specializedcuvettes. However <strong>with</strong> recent innovations of theNucleofector Technology some cell types can now betransfected in adherence (see page 152).An Important Note about Cancer Cells and NucleofectionFor the Nucleofection of cells from solid tumors, a primary cell kit canbe used if the cells are less than passage 3. If the cells are passage 3 orhigher, we recommend using a Cell Line Nucleofector Kit. For suspensioncells, like leukemic cells, if the cells are less than passage 5, aNucleofector Kit for primary cells can be used. If the cells are passage5 or higher, we recommend using a Cell Line Nucleofector Kit.Suspension cells – Suspension cells should be transfectedwhen they are in the logarithmic growth phase. Generally,this corresponds to a density of 2–5 × 10 5 cells per ml. ForNucleofection of some cell types, a higher density isrecommended. Please check the Optimized Protocol for thecell type you are using. The cells should be passaged 2–4days prior to transfection so that they achieve the requireddensity for your experiments.For both adherent and suspension cells, it is important tomake sure that the culture is growing properly and that thecells have the proper morphology. If they do not, this couldindicate contamination <strong>with</strong>, for example, bacteria, fungi, ormycoplasma. Mycoplasma are common contaminants ofcells grown in culture; studies indicate that between 5 and35% of cultures are contaminated. Infections, which lead tomany serious alterations in cell function and geneexpression, are persistent and difficult to detect usingconventional methods. We recommend our convenient20-minute luminescent mycoplasma test – MycoAlertMycoplasma Detection Kit (Cat. No. LT07-118; see page139). If mycoplasma are detected, we strongly recommenddiscarding the cells. If the cells are truly irreplaceable,MycoZap Mycoplasma Elimination Reagent is a gentle,effective option (Cat. No. LT07-818; see page 142).www.lonza.com/mycoplasmaCell HarvestingProper cell handling during the harvesting process is crucialin order to maintain the health of the cell and helps ensurethe success of your experiments. Before harvesting yourcells, we recommend washing the monolayer to get rid ofany residual growth medium, as well as calcium andmagnesium ions. In most cases, PBS or HBSS <strong>with</strong>outcalcium or magnesium can be used. Other wash solutionscan be used as well and will depend on the characteristicsof the cell in use. For example, cultures that have multiplelayers may detach easier if they are washed first <strong>with</strong> a0.5 mM –1 mM EDTA solution or trypsin solution.For Nucleofection in suspension, adherent cells will needto be detached from the culture vessel before they can betransfected. For cell lines, we recommend using trypsin at aconcentration of 0.05% (0.5 mg/ml) and EDTA at 0.48 mM(0.2 mg/ml) in a balanced salt solution <strong>with</strong>out calcium andmagnesium.Technical Information / Transfection9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com413
Cell Culture Tips for Cell Lines and Primary Cells Prior to TransfectionContinuedTechnical Information / TransfectionPrimary cells, like Clonetics Cells, should be treated moregently than cell lines. For example, we recommend usingthe Clonetics ReagentPack (CC-5034; see page 10), whichcontains a gentle trypsin solution, HEPES buffered saline,and trypsin neutralizing solution. Other dissociativeenzymes can be used and again, this depends on thecharacteristics of the cell. For example, collagen richcultures may require a collagenase dissociation. Pleasecheck <strong>with</strong> the cell supplier for specific recommendations.Once the cells have detached from the growth vessel,inactivate the trypsin by adding either:––Growth medium <strong>with</strong> serum––Trypsin neutralizing solution from ReagentPack(CC-5034; see page 10)––PBS/0.5% BSAIn all cases, it is important to monitor the cells duringtrypsin treatment because, if the trypsin is allowed toremain on the cells longer than necessary, it will damagethe cell membranes resulting in high cell mortality.Do not scrape the monolayer unless specificallyrecommended by the cell supplier. Scraping can causemechanical damage to the cells and will not result in asingle cell suspension.When using biochemical transfection reagents fortransfection, it is not necessary to detach the cells from thegrowth vessel.When working <strong>with</strong> suspension cells, no detachment isnecessary; simply spin down the required number of cellsand remove as much residual growth medium as possiblefrom the cell pellet. For Nucleofection, the cells will then beresuspended in Nucleofector Solution.It is also important to avoid extra pipetting or unnecessarywashing steps. Do not vortex your cells, as extra handlingbeyond what is recommended can potentially harm thecells and result in high cell mortality.An Important Note About Lipids and TransfectionMost lipids cannot be used on suspension cells. It is important not to addantibiotics to the medium during transfection <strong>with</strong> lipids, as this will causecell death. Antibiotics can be added to the growth medium aftertransfection. In some cases, it may be helpful to allow the cells to recoverfor 12–24 hours before adding any antibiotics.Serum can be present in the growth medium after transfection and, insome cases, it can also be present in the transfection medium. Serummust not be present during complex formation because it inhibits theformation of the liposome complexes.With lipids, it is also possible to scale up the transfection by varying theamounts of lipid, DNA, cells, and medium in proportion to the relativesurface area of the culture vessel. Please check the protocol specific toyour lipid for guidelines.An Important Note About CentrifugationFor Nucleofection, it is important to follow the centrifugation guidelinesas stated in the Optimized Protocols. Our standard for centrifugation is90xg. We do not use RPM’s because the speed you select in order toachieve 90xg will vary <strong>with</strong> the type of rotor in use in your lab. In orderto determine the required speed to get 90xg, please consult the operationmanual for your centrifuge or rotor.If you do not have the manual, please visit the following link for anomogram that you can use to convert g-forces to RPM: http://aquaticpath.umd.edu/nomogram.html.Alternatively, the correct rotor speed can be calculated by measuringthe maximum radius of your rotor and entering the information into thetable found on the Brinkmann website atwww.sciencegateway.org/tools/rotorCentrifugation speeds and g-forces are not as critical <strong>with</strong> othertransfection methods (i.e., standard electroporation, lipids) as they are<strong>with</strong> Nucleofection, which is why you will not see specific guidelinesgiven in many protocols.Cell SourcesFor best transfection results, we recommend the use ofcells <strong>with</strong> a known history, i.e., low passage number andfree from contamination.For primary cells, we recommend using Clonetics andPoietics Cells from Lonza.9Helpful Hints for Adherent CellsIf you are working <strong>with</strong> a strongly adherent cell line, you can use a strongertrypsin solution. Solutions of 0.25% and 0.5% trypsin are routinelyavailable from commercial suppliers. Alternatively, instead of washingthe monolayer <strong>with</strong> PBS before the addition of trypsin, you can use atrypsin solution as the wash. Aspirate the trypsin, replace <strong>with</strong> freshtrypsin solution, and incubate until the cells detach.If the cells are weakly adherent, you can wash <strong>with</strong> EDTA alone, whichmay be enough to detach the cells or you can try the following: Wash cells<strong>with</strong> PBS, add trypsin and immediately aspirate off the trypsin solution.Incubate the cells <strong>with</strong> the residual solution until they detach.Alternatively, you can also wash the cells <strong>with</strong> ice cold PBS if the cells areparticularly loose. In many cases, this alone may cause them to detach..414North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Important Vector Factors for Gene ExpressionIntroductionOur Scientific Support Team is commonly asked: “Why don’tI get the same expression level of my gene if I use variousvector backbones?" There are many components to a vectorthat can have an effect on the level of gene expression.Below you will find the 10 most important factors to considerwhen looking at vectors.The selection of an appropriate expression vector is crucialfor efficient gene expression. Just take a look at Figure 1.We tried 10 different vectors expressing the same luciferasegene in different backbones and expression cassettes andobtained highly variable expression levels.Relative luciferase-expression % of[pGL3-CMV at 24 h]A1600140012001000800600400■■Promoter StrengthIs your promoter appropriate for the cell type that youare working <strong>with</strong>? Table 1 describes the promoterstrengths as a relative percent of the strength of theCMV promoter for various cells for which we haveOptimized Protocols. The CMV promoter activity is set to100% based on the CAT assay values from the referencedpublications. Although CMV is a strong promoter in manymammalian cells, another promoter may give strongerexpression in your cells (e.g., promoter SV40 in BHK-21cells).■■IntronsMany researchers consider constitutively splicedintrons to be required for optimal gene expression;however, this point is not always agreed upon. Theintron position and strength can affect transcription,mRNA export and polyadenylation. Thus, depending onits position, an intron can even lead to decreased geneexpression.Technical Information / Transfection200Relative luciferase-expression % of[pGL3-CMV at 24 h]0B2000150010005000ControlControlProgramonlyProgramonlypGL3-controlpcDNA3.1 pFP pIRES- pcDNA4MCS B HisMaxpGL3-controlpGL3-CMVpGL3-CMVpmaxCloningpmaxCloningpSG5pSG54 hours 24 hours 48 hourspcDNA3.1 pFP pIRES- pcDNA4MCS B HisMaxpCMVbetapCMVbetapCMV-TNTpCMV-TNTFigure 1. Luciferase expression levels depend on vector backbones. We looked at luciferase expression at 4, 24 and 48 hours in THP-1 and HUVEC cells. Theamount of DNA was held at equimolar amounts based on plasmid size. For THP-1, the DNA amount ranged from 0.3–0.5 μg per reaction (A). For HUVECs, we used2.5–4.4 μg of plasmid (B).9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com415
Important Vector Factors for Gene ExpressionContinuedTechnical Information / TransfectionTable 1: Promoter Strengths in Different Cell TypesCell Line Source of Cells SV40 EF1a RSV CMV Reference293 Human embryonic kidney 5% 74% 100% 4, 6BHK-21 Hamster kidney 200% 200% 100% 4C6 Rat glioma 44% 100% 5CHO-K1 Chinese hamster ovary 16% 11% 100% 2, 4, 5Cos-7 African green monkey kidney 7% 15% 7% 100% 1, 2, 4, 5, 8HeLa Human cervical carcinoma 43% 73% 29% 100% 2, 3, 4, 5, 6, 8N2A (Neuro-2A) Murine neuroblastoma 50% 100% 5NIH-3T3 Murine fibroblast 67% 143% 107% 100% 2, 3, 7, 8■■IRES PlasmidsWith IRES plasmids, the promoter drives the expressionof two genes, the cloned gene of interest (usually theupstream gene), and a reporter gene, often encoding aGFP protein (the downstream gene). The mRNAexpressed from an IRES plasmid is a bicistronicmessage, meaning that both genes are present on thesame mRNA molecule. Equal amounts of the messageswhich encode each gene are present in the mRNApopulation. However, the translation initiation efficiencyof the two genes differs significantly. Ribosome bindingto the initiation region of the upstream gene is veryefficient, while the IRES allows ribosome binding andtranslation initiation for the downstream gene often at asignificantly lower level. Since the downstream gene isusually GFP, the expression level of GFP will be lowerthan it would be normally seen when compared toplasmids <strong>with</strong>out an IRES-sequence. Equal amounts ofthe protein of interest and GFP can be obtained using afusion of both proteins, constructs containing twoexpression cassettes, or co-transfection.We looked at pIRES expression vectors (Clontech) <strong>with</strong>a reporter (GFP) cloned in either the Multiple CloningSite (MCS) upstream of the IRES (construct A) ordownstream of the IRES (construct B; Figure 2).ApmaxGFP Vector:CMV*pmaxGFP VectorConstruct A: CMV*pmaxGFP Vector IRESMCS B emptyConstruct B: CMV*MCS B empty IRESpmaxGFP Vector9BExpression in HL-60Mean fluorescence (% of pmaxGFP Vector)12010080604020Expression in HUVECMean fluorescence (% of pmaxGFP Vector)120100806040200pmaxGFP Vector Construct A Construct B0pmaxGFP Vector Construct A Construct BFigure 2. Reporter gene expression is dependent on the position in an IRES expression vector. HL-60 and HUVEC cells were transfected by Nucleofection <strong>with</strong>either pmaxGFP Vector or pIRES variants containing maxGFP Reporter Protein cloned in either the Multiple Cloning Site (MCS) upstream (construct A) ordownstream of the IRES sequence (construct B; 2A). Figure (2B) shows reduced GFP expression using IRES plasmids especially if GFP is located downstreamof the IRES sequence.416North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Important Vector Factors for Gene ExpressionContinuedFigure (2B) demonstrates that GFP expression isdrastically reduced if GFP is located downstream of theIRES. This study is shown for the GFP reporter gene, butwas also done using luciferase <strong>with</strong> similar results.Does your plasmid contain an IRES sequence? If so,where is it located? Keep in mind that the stability of thebicistronic mRNA can be influenced by either of theinserted genes. The levels of expressed protein for thefirst and second genes will not be identical, and this cancreate problems <strong>with</strong> analysis and interpretation. As aresult, the true efficiency of the plasmid can beunderestimated due to the lower expression level ofyour reporter. Be sure to use a very sensitive detectionmethod for the reporter gene down stream of the IRES.■■LTR (Viral Long Terminal Repeats)Some expression plasmids utilize promoters andenhancers obtained from the Long Terminal Repeats(LTRs) of retroviruses, and when these expressionplasmids are transfected into certain cells, theexpression of the cloned genes might be suppressed bythe cell. Although the mechanism of suppression is notcompletely understood, it is likely that plasmidscontaining promoters or enhancers derived fromretroviral LTRs will not function well in primary cells andsome cell lines. As Nucleofection is often the onlyeffective method for primary cells, such suppressiveeffects might be observed more frequently <strong>with</strong>Nucleofection. The only effective alternative can be toreclone the gene of interest into a different expressionplasmid that uses conventional promoters such as CMV(e.g., pmaxCloning Vector), EF1a or SV-40.Kinetics of Luciferase Expression in HUVECRelative expression per cell (% of 24 hours value)c f L c n3002502001501005006 – 16 hours10 20 30 40 50Time post-transfection (hours)■■Size of VectorWe routinely use plasmids of 4–7 kb in our laboratoriesand Nucleofection of plasmids up to approximately 20kb can be achieved. Using plasmids larger than this willmost likely result in lower transfection efficiency. Thegeneral rule is that the larger a plasmid, the moredifficult it becomes to get it inside the cell. This is trueforelectroporation or lipid-mediated transfectionsNevertheles, some preliminary results usingNucleofection indicate that BAC’s can be transfected as9, 10well but also <strong>with</strong> low transfection efficiency.■■ReporterWhat kind of reporter are you using? It needs to be safe,reproducible, quantitative, and sensitive. Your reportershould not be expressed by the cell endogenously athigh levels and should function well <strong>with</strong> yourdownstream assays. When you change reporters, or ifyou change transfection methods, the kinetics of thatreporter’s expression using the current transfectionmethod need to be evaluated to make sure that you areanalyzing at the optimal time point. Luciferase, forexample, has very different expression kinetics,depending on whether the transfections are being doneby Nucleofection (maximum expression at 6–16 hourspost-transfection) or lipids (maximum expression at 24hours post-transfection). We found that luciferasekinetics are related to the transfection method and notto the vector backbone or cell type tested. Since kineticsof reporter expression also depend on mRNA andprotein stability, we then compared the kinetics ofluciferase expression to that of ß–gal expression(Kinetics of ß-gal Expression in HUVECRelative expression per cell (% of 24 hours value)30025020015010050010 – 48 hours10 20 30 40 50Time post-transfection (hours)Technical Information / Transfection9Figure 3. Expression kinetics are reporter gene dependent. HUVEC cells were transfected by Nucleofection <strong>with</strong> either a luciferase or a ß-gal expression vector.While luciferase expression shows a maximum 6–16 hours post-transfection, ß-gal expression is sustained for several days after transfection. Values representrelative protein amounts per well, normalized to 24 h value set to 100%.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com417
Important Vector Factors for Gene ExpressionContinuedTechnical Information / Transfectionfollowing Nucleofection (Figure 3). These data arefrom a co-transfection of HUVEC cells <strong>with</strong> a luciferasevector and a ß–gal vector. Both reporters can be underrepresentedat very high levels. However, the kineticsof expression for each reporter are very different.Luciferase has a very pronounced drop-off of expressionafter 16 hours. However, the ß–gal expression reachesa maximum at 10 hours post-transfection and levelsoff. If a single time point for analysis is chosen, such as24 hours, the maximum expression of luciferase will bemissed and again can under-represent the efficiency ofthat vector. As a consequence, we recommendperforming luciferase analysis 6–16 hours postNucleofection, whereas the optimal analysis timepoint for ß–gal or GFP expression is after 10–48 hourspost Nucleofection.■■Detection MethodsThe detection method is predetermined by the reporter.Some reporters can be measured in multiple ways. GFP,for example, can be read by a fluorescent microscope,flow cytometer or a fluorescent plate reader. If only aqualitative picture is needed, the fluorescentmicroscope can provide a cost effective option. Whenlooking for quantitative data, a flow cytometer orfluorescent plate reader should give more accuratedata.■■Fusion Vectors vs. Co-TransfectionsExpression of a fusion protein depends on thelocalization of the protein, transcription and translation,as well as the folding and stability of the fusion protein.To improve expression, it may also be advisable tochange the terminus to which the protein is fused.Co-transfections can be used instead of a fusion vector.One plasmid would contain the reporter gene (i.e., GFP)and the second plasmid would contain the gene ofinterest to be expressed. Depending on differences inpromoter strength and vector size, the ratio of the twovectors needs to be optimized.■■Hairpin Structures in Gene ProductA hairpin structure that forms in the RNA can affecttranslation of the gene. This should be considered, forexample, when introducing mutations into the gene tobe expressed.■■Kozak SequenceThe Kozak sequence can slow down the rate of scanningby the ribosome and improve the chance of itrecognizing the start of translation at the ATG startcodon. If the Kozak sequence is contiguous <strong>with</strong> the ATGstart codon, it can greatly increase the efficiency oftranslation and the overall expression of the gene ofinterest.9References1. Cheng et al., (1995) Int J Radiat Biol 67(3): 261-2672. Foecking et al., (1986) Gene 45(1): 101-1053. Davis et al.., (1988) Biotechnol Appl Biochem 10(1): 6-124. Liu et al., (1997) Anal Biochem 246(1): 150-1525. Wenger et al., (1994) Anal Biochem 221(2): 416-4186. Kronman et al., (1992) Gene 121(2): 295-3047. Thompson et al., (1993) In Vitro Cell Dev Biol 29A(2): 165-1708. Thompson et al., (1990)Gene 96(2): 257-2629. Marshall et al., (2005) J Biol Chem 280(39): 33357-3336710. Wang et al., (2006) J Virol 80(12): 6003-6012418North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Essentials for Preparing a Transfection Experiment <strong>with</strong> Plasmid DNAPreparation and QualityThe quality of DNA used for transfection plays a central rolefor the success of the experiment.We strongly recommend the use of high quality products forplasmid purification, e.g., Qiagen® EndoFree® Plasmid Kits.The purified DNA should be resuspended in sterile deionizedwater or TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0)before use. It has been demonstrated that DNA which wasnot purified via an endotoxin-free method can result in poorcell viability for some cell types. The same effect can beobserved when endotoxin is added to clean DNApreparations. We do not recommend using phenol:chloroformor other organics in the preparation of the DNA as these aretoxic to living cells and very difficult to remove completely.For cells which are sensitive to activation bylipopolysaccharides, such as monocytes, macrophages anddendritic cells, an additional purification step via PEGprecipitation is helpful. DNA should be dissolved in water. To100 μl DNA in water, add 750 μl 5.0 M NaCl and 750 μl 40%w/v PEG 8000. Mix the contents of the tube by invertingseveral times and incubate on ice for one hour. Spin at topspeed in a microcentrifuge for 15 minutes at 4°C. Removesupernatant, dissolve pellet in 100 μl water and repeat PEGprecipitation. Carefully remove supernatant. Rinse the pellet<strong>with</strong> 500 μl ice cold 70% ethanol. Spin 3 minutes. Removesupernatant. Air dry the pellet and resuspend in 20 μl sterilewater or TE (adapted from Molecular Cloning: A LaboratoryManual (Third Edition) by Joseph Sambrook, PeterMacCallum Cancer Institute, Melbourne, Australia; DavidRussell, University of Texas Southwestern Medical Center,Dallas).Measuring Quality and Concentration of DNADNA purity should be measured by the ratio of absorbance(A) at 260 and 280 nm. The A260/A280 ratio should be at orabove 1.6 for transfection use. Additionally, the plasmidshould be run on an agarose gel to check for any nicked DNAor degradation. At least 90% of the DNA should be in thesupercoiled conformation and no degradation productsshould be visible. To determine concentration, measure theabsorbance at a wavelength of 260 then calculate asfollows:A260 × 50 μg/ml × dilution factor = DNA concentration.Be sure that the dilution used is in the linear range of thespectrophotometer, usually an OD of 0.1–1.0. If using amicrocuvette <strong>with</strong> a path length of less than 1 cm, it will benecessary to multiply by the factor to convert to the OD of a1 cm path. For example, the path length of the 5 μl cuvette isonly 0.5 mm or 1/20 cm, so it would require multiplying theabove formula by 20 to get the concentration.Optimal DNA Amounts for NucleofectionGene transfer efficiency can also be affected by theamounts of DNA. For Nucleofection of most cell types, westart <strong>with</strong> 1–2 μg DNA per 100 μl reaction <strong>with</strong> our pmaxGFPVector which is ~4 kb in size. For larger constructs, it may benecessary to add higher amounts of DNA, so we recommendtitrating the DNA to see if increasing amounts are helpful.The plasmid amounts can be increased up to 10 μg persample or more in some cases. However, certain cells aresensitive to DNA and, in those cases, more DNA will result inincreased mortality of the cells. If the Optimized Protocol fora cell type recommends using less than 2 μg of pmaxGFPVector, those cells are likely DNA sensitive.NOTE: The DNA concentration should be such that no more than 10 μl ofsubstrate per 100 μl reaction is added in order to not dilute the NucleofectorSolution too far or exceed the tolerance of the cuvette, which could result inan error on the device.Working <strong>with</strong> Highly Diluted DNAIn order to keep the total DNA volume to add to aNucleofection in the appropriate range, it may be necessaryto ethanol precipitate your DNA if it is too diluted. Anammonium acetate-based ethanol precipitation followed bytwo 70% ethanol washes should ensure that there isminimal salt carry over. The procedure is to add 0.5 volumesof 7.5 M ammonium acetate and 2 volumes ethanol to theDNA in solution and mix well. Spin at full speed in amicrocentrifuge for 15 minutes. Carefully removesupernatant. Rinse the pellet <strong>with</strong> a volume equal to theprecipitation of ice cold 70% ethanol. Spin for 5 minutes.Remove supernatant. Repeat. Air dry the pellet andresuspend in sterile water or TE. Generally an assumption ofabout 70% recovery is good for determining the volume toresuspend. Then read A260 to confirm.Technical Information / Transfection9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com419
Guideline for Generation of Stable Cell LinesTechnical Information / TransfectionBackgroundStable, long-term expression of a gene of interest can beeither achieved by eukaryotic vectors that harbor elementsfor episomal maintenance in the nucleus of a transfectedcell or via direct integration of the transfected plasmid intothe target cells genome. Episomal stability is often limited,resulting in gradual loss of transfected vectors that canonly be prevented by sustained antibiotic selectioneliminating cells that lost the plasmid. Furthermore, thefunctionality of episomal plasmid elements is oftenrestricted to certain species. Although integration into thehost cell chromosome is a rare event and, for mostpurposes, clonal events have to be isolated, stability of theintended genetic modification usually is much higher.Initially, the gene of interest has to be introduced into thecell (A), subsequently into the nucleus (B), and finally, ithas to be integrated into chromosomal DNA (C) (Figure 1).Since chromosomal integration into host chromosomes is arare event, stably-transfected cells usually have to beselected and cultured in various ways. For the selection ofstably-transfected cells, a selection marker is co-expressedon either the same construct or on a second, co-transfectedvector. A variety of systems for selecting transfected cellsexists, including resistance to antibiotics, such as neomycinphosphotransferase, conferring resistance to G418,dihydrofolate reductase (DHFR), or glutamine synthetase(Southern and Berg, 1982). After gene transfer, cells arecultivated in medium containing the selective agent. Onlythose cells which have integrated the plasmid containingthe drug resistant gene survive.Several options are used for the generation of a stablecell line, depending on the scope of the experiment (seeTable 1). A mixed population of drug resistant cells can beused directly for experimental analysis (batch culture) <strong>with</strong>the advantage of generating fast results, but also thedisadvantage of dealing <strong>with</strong> an undefined and geneticallymixed cell population. To generate clonal cells, it isnecessary to dilute the resistant cells in such a way thatculture as single, isolated cells is achieved e.g., by plating in96-well plates or other methods. Subsequently, theselection process is applied to the single cell cultures. Theprocedure of single cell cloning may be repeated severaltimes to obtain 100% clonal purity. This culture methodallows for conduction of the study or the screening using adefined and homogenous cell system. So far, generation ofstable cell lines has been a major challenge for many celltypes (e.g., Jurkat, MCF7 or U937) since overall transfectionefficiencies and/or integration frequencies have been low.While common transfection methods, such as lipofection,can be used for the stable expression in easy-to-transfectcell lines (e.g., HeLa, COS-7 or CHO), Nucleofection is themethod of choice for stable expression in difficult-totransfectcell types.The generation of stably-transfected cell lines is essentialfor a wide range of applications, such as gene functionstudies (Grimm, 2004), drug discovery assays or theproduction of recombinant proteins (Wurm, 2004). Incontrast to transient expression, stable expression allowslong term, as well as defined and reproducible, expressionof the gene of interest.9BCAFigure 1. The general path of stable integration.420North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Guideline for Generation of Stable Cell LinesContinuedTable 1: Different Strategies for Stable Clone GenerationCulture System Advantage ApplicationBatch culture – polyclonalFast, useful for cells which do not grow insingle cell cultureIn a batch culture system, a mixed population of drug resistant cells is selected on plates or in flasks and can be used directly for experimental analysis. Duringa limiting dilution procedure, cells are usually diluted and selected e.g., in a 96-well plate for outgrowth of cell clones or single colony growth. Subsequently,colonies can be picked and used to generate monoclonal cell lines.Culture Conditions for Generation of Stable Cell LinesAs for transient transfection experiments, culture conditions(passage number, split rhythm, etc.) of your selected celltype are very important for the generation of stablytransfectedcell lines. For optimal results, we recommendusing the cell culture recommendations of the supplier forthe respective cell type. In general, the cell line should bepassaged two days before the experiment to promote goodproliferation and cell physiology. Cell passage should not behigher than 30. Interference of higher passage numbers<strong>with</strong> integration efficiency is possible and may be cell-typedependent.Depending on the scope of your experiment, cells can becultivated as polyclonal batches or monoclonal single cellclones post transfection.Overexpression, protein expression systems(e.g., for basic research)Limiting dilution – monoclonal Defined cell clones Studies of gene function, protein production(e.g., for therapeutic applications)Transfection MethodStable expression can be influenced by the transfectionmethod used. The choice of transfection method determineswhich cell type can be targeted for stable integration. Whilebiochemical transfection reagents can be used to transferDNA into standard cell lines, efficient delivery of DNA intonotoriously difficult-to-transfect suspension cell lines oreven primary cells is only possible <strong>with</strong> viral methods orNucleofection (Figure 2). Unfortunately, viral methodssuffer from several limitations, such as time consumingproduction of vectors and safety concerns (Hacein-Bey-Abina et al., 2003).We recommend using the Nucleofector Technology (seepage 168), for transfection of difficult-to-transfect cell lines.Technical Information / TransfectionIntegration frequency [%]0.160.140.120.100.080.060.040.0290.00Linear DNA Circular DNA 10 DNA µg circular DNANucleofectionLipofectionFigure 2. Higher integration rates in difficult-to-transfect cell lines usingNucleofection. Jurkat cells were transfected using either Nucleofection (2μg DNA) or lipofection Reagent L (0.7 μg DNA) according to the respectivemanufacturer’s instructions. 24 hours after transfection, cells were platedon a 96-well plate containing culture medium supplemented <strong>with</strong> G418for selection of stably-transfected cells. 30 days after plating, cells wereanalyzed for clonal outgrowth (Integration frequency = number of resistantclones per number of living cells seeded). Due to toxic effects, lipofection<strong>with</strong> 10 μg circular DNA could not be performed.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com421
Guideline for Generation of Stable Cell LinesContinuedTechnical Information / TransfectionExperimental OutlineExpression Plasmid Procedure Outline Important InformationStep Expression 1 plasmidStep 2Design experiment and choosecell type, expression vector andtransfection method.Determine appropriate cell numberper well (only for limiting dilution)and G418 concentration.Make sure that transfection methodand expression vector are suitable foryour cell type.Cells differ in their susceptibility to G418.The active concentration of stock G418 canvary from batch to batch.Step 3 Expression plasmid + cellsTransfect expression vector into cells. Amount of expression vector perexperiment is dependent ontransfection method and cell type.Step 4Plate transfected cells and cultivatecells in medium <strong>with</strong>out G418.Do not add G418 to culture mediumimmediately after transfection as thismay drastically increase mortality.Step 5Dilute cells into culture plates andstart selection 24–48 hours post-transfection.Feed every 2–3 days (for batchculture) or 10 days (for limitingdilution) <strong>with</strong> selection medium.Choose culture conditions (batchculture, limiting dilution) dependingon your experimental design.Refreshed selection medium isimportant to avoid false positive cells.9Step 6 Analyze stably transfected cells. Make sure that the chosen assayis suitable for your application.422North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Guideline for Generation of Stable Cell LinesContinuedProtocol for Batch Culture■■Determination of G418 Concentration Using BatchCultureStably-transfected cells can be selected by the additionof drugs to the culture medium, if the expressionplasmid carries a drug resistance gene. Here wedescribe the neomycin resistance system, which usesresistance to G418 as a selection marker. Cells differ intheir susceptibility to G418, which may even vary <strong>with</strong>cell passage numbers. Cells that are cultured in serumfreemedia may require much lower G418 concentrationsas compared to cells in media containing sera. Theselection condition for your specific cell type needs tobe established experimentally. Determine the minimumlevel of G418 to be added to the culture medium toprevent cell growth. Note that the active concentrationof stock G418 can vary considerably from batch tobatch. We therefore recommend testing G418 sensitivityfor every new batch or to buy a large amount of one lotto standardize selection conditions.1. Split cells into 12-well plates containing culture medium<strong>with</strong>out G418 in plating densities according to cellsupplier instructions.2. The next day, aspirate growth medium and feed cells<strong>with</strong> medium containing increasing concentrations ofG418 (e.g., titrate G418 in a range of 0.1 mg/ml to3. 1.5 mg/ml; in serum-free culture expand range down to20 μg/ml).4. Feed cells every 2–3 days <strong>with</strong> selection medium.5. Check cell death after 7–14 days by light microscopy.6. Choose the concentration which is 0.1 or 0.2 mg/mlabove the one which shows complete cell death as theappropriate G418 concentration for selection. If thelowest concentration used shows complete cell deathat day 7, the titration should be repeated <strong>with</strong> a lowerconcentration range.■■TransfectionFor transfection, please follow the respectivemanufacturer’s instructions of your transfectionsystem and transfect the expression plasmid,containing the gene of interest and the sequence for adrug resistance gene, into your cell type. Aftertransfection, plate cells according to the instructionsfrom the supplier of your transfection system on tissueculture plates. Usually 6-well plates are used for 10 6adherent cells and 12-well plates for 10 6 suspensioncells.Important controls:We suggest including a sample of untransfected cells as a negativecontrol for selection. We also strongly recommend checking thetransfection efficiency and integration frequency of your experiment <strong>with</strong>a GFP-control plasmid, such as pmaxFP-Green Vector. pmaxFP-GreenVector encodes the green fluorescent protein (maxFP-Green ReporterProtein) from Pontellina. pmaxFP-Green Reporter Protein expressingcells can easily be analyzed by fluorescence microscopy or flowcytometry to monitor the efficiency of your experiment.Cell Culture Post TransfectionUnder selective conditions, resistant cells outgrownon-resistant cells, resulting in a polyclonal population ofstably-expressing cells. This heterogeneously expressingpopulation of resistant cells can then be used forexperimental analysis.1. After transfection, allow cells to grow and to expressthe protein for G418 resistance under non-selectiveconditions for at least 24 hours (for sensitive cells,G418 selection may begin after 48 hours).2. Trypsinize adherent cells by standard procedures oruse suspension cells directly for analysis. If possible,analyze for transfection efficiency 24–48 hours posttransfectionon an aliquot of the positive control sampleand your gene of interest (transient transfectioncontrol).3. For the selection of stably expressing cells, cultivatecells in standard medium <strong>with</strong> supplements and theappropriate amount of G418, pre-tested for your celltype.4. Plate cells on culture plates or flasks according to cellsupplier instructions and incubate cells under standardconditions.5. Feed and, if necessary, split cells until outgrowth ofresistant cells.6. Harvest cells of batch culture.Usually, cells which have not integrated the resistancegene die during the first days of selection. Outgrowth ofresistant cells can be observed normally after 2 weeks ofselection. For some cells this may take up to 4 weeks. G418is labile at 37°C, therefore it is recommended to changemedium containing G418 every 2–3 days to compensatefor loss of selection pressure. In some cases, it might befeasible to lower the G418 concentration after 1–2 weeks.Cells should be grown for at least 3 weeks under selectionpressure to avoid contamination <strong>with</strong> non-resistant cells.Negative control wells (e.g., sample <strong>with</strong>out expressionplasmid) should be inspected by light microscopy andshould not contain any signs of cell growth. Dependent oncell type and cell growth, selection can be extended up to4–5 weeks or longer in total.Technical Information / Transfection9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com423
Guideline for Generation of Stable Cell LinesContinuedTechnical Information / Transfection9■■Analysis of Batch CultureOnce you have obtained resistant cell batches or clones,expand the cells and assay for your gene of interest. Forthe analysis of maxFP-Green Reporter Proteinexpressing cells (positive control), use fluorescencemicroscopy or flow cytometry.Protocol for Limiting Dilution■■Determination of G418 Concentration and PlatingDensityStably-transfected cells can be selected by the additionof drugs to the culture medium, if the expressionplasmid carries a drug resistance gene. Here wedescribe the neomycin resistance system, which usesresistance to G418 as a selection marker. Cells differ intheir susceptibility to G418, which may even vary <strong>with</strong>cell passage numbers. Cells that are cultured in serumfreemedia may require much lower G418 concentrationsas compared to cells in media containing sera. Theselection condition for your specific cell type needs tobe established experimentally. Determine the minimumlevel of G418 to be added to the culture medium toprevent cell growth. Note that the active concentrationof stock G418 can vary considerably from batch tobatch. We therefore recommend testing G418sensitivity for every new batch or buying a large amountof one lot to standardize selection conditions. The finalplating density after transfection depends on theculture conditions of the specific cell type and the G418concentration. We therefore recommend combining thetitration of G418 <strong>with</strong> the titration of cell numbers fordetermination of plating density in a matrix (Figure 3).1.4 mg/mlADecreasing cell numbers2000100050025012563321612 3 4 5 6 7 8 9 10 11 1284211. Pre-plate 100 μl medium in each well of the plate.2. Add 100 μl of cell suspension containing 4000 cells perwell to the first column (#1).3. Carry over 100 μl to the next column after gentle up anddown pipetting, thereby diluting in a ratio of 1:2. Repeatthis procedure for each consecutive column.4. After completing, discard 100 μl from the last column(#12). The first column should then contain about2,000 cells, the last column less than one cell onaverage.5. Add 100 μl of G418 containing medium (2.8 mg6. G418 per ml) to the first row (A) for a final G418concentration of 1.4 mg/ml.7. Add G418 to the following rows in decreasingconcentrations of G418 in steps of 0.2 mg/ml. For thelast row (H) add medium <strong>with</strong>out G418.8. Incubate cells at standard conditions.9. Analyze cell growth by microscope. In some cases, cellgrowth can also be observed by change of mediumcolor.10. If you observe cell growth (after >10 days) in the wells<strong>with</strong>out G418 containing less than 4 cells, it isreasonable to assume that those cells can grow outstarting as single cells.11. Choose the G418 concentration which is just above theone which shows complete cell death as the appropriateG418 concentration for selection.Certain cell types might need a critical number ofneighboring cells to grow appropriately. If this is the case,limiting dilution experiments can only be done at higher cellconcentrations, making it more difficult to obtain pureclones. In these cases, clones should be generated byplating an appropriate number of cells, selecting anddiluting the resistant clones together <strong>with</strong> non-transfectedcells of the same type as feeder cells. Other possibilities areculturing cells in 96-well plates <strong>with</strong> communicatingchannels, on soft-agar or methylcellulose.Decreasing G418 concentration1.2 mg/ml1.0 mg/ml0.8 mg/ml0.6 mg/ml0.4 mg/ml0.2 mg/ml0 mg/mlBCDEFGHFigure 3. Matrix titration of G418 and titration of cell number fordetermination of plating density in one 96-well plate.424North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Guideline for Generation of Stable Cell LinesContinued■■TransfectionFor transfection, please follow the respectivemanufacturer’s instructions of your transfectionsystem and transfect the expression plasmidcontaining the gene of interest and the sequence for adrug resistance gene into your cell type.After transfection, plate cells according to theinstructions from the supplier of your transfectionsystem (e.g., on 96-well tissue culture well plates).Important controls:We suggest including a sample of untransfected cells as a negativecontrol for selection. We also strongly recommend checking thetransfection efficiency and integration frequency of your experiment<strong>with</strong> a GFP-control plasmid, such as pmaxFP-Green Vector. pmaxFP-Green Vector encodes the green fluorescent protein (maxFP-Green)from Pontellina p. maxFP-Green Reporter Protein expressing cellscan easily be analyzed by fluorescence microscopy or flowcytometry to monitor the efficiency of your experiment.■■Cell Culture Post-transfectionSingle cell clones from adherent and suspension celltypes can be generated by diluting cells in a 96-wellplate or other methods that allow the outgrowth ofisolated cell clones under selective pressure.1. After transfection, allow cells to grow and to express theprotein for G418 resistance under non-selectiveconditions for at least 24 hours (for sensitive cells,G418 selection may begin after 48 hours).2. Trypsinize adherent cells by standard procedures oruse suspension cells directly for analysis. If possible,analyze for transfection efficiency 24–48 hours posttransfectionon an aliquot of the positive control sampleand your gene of interest (transient transfectioncontrol).3. Count living cells via trypan blue staining or otherappropriate methods.4. Using standard medium <strong>with</strong> supplements and theappropriate amount of G418 pretested for your celltype, plate cells in a 96-well plate <strong>with</strong> different cellnumbers per well (e.g., 10, 100, 1000) in a volume of atleast 100 μl per well. Depending on cell concentrationdetermined before, conduct several serial dilution stepsas applicable. It is important to thoroughly suspendcells before seeding, but avoid harsh treatment byfrequent pipetting. Use the lower limit determinedbefore as the minimum number of cells per well (forgeneration of single cell clones, choose the dilutionwhich statistically yields between 5 and 20 clones per96-well plate, thereby minimizing the probability ofwells <strong>with</strong> more than one clone).5. Incubate cells under standard conditions and feed cellsafter 10–14 days <strong>with</strong> fresh selection medium.6. Cell clones can be analyzed or further expanded assoon as cells in the non-transfected control wells havecompletely died.7. In order to help assure that selected cell populationsrepresent clones from a single cell, another round oflimiting dilution under selection is recommended.Usually, cells which have not integrated the resistancegene die during the first days of selection. Outgrowth ofresistant cells can be observed normally after 2 weeksof selection. For some cells, this may take up to 4 weeks.G418 is labile at 37°C, therefore, it is recommended toadd fresh medium containing G418 after 10–14 days tocompensate for loss of selection pressure. In somecases, it might be feasible to lower the G418concentration after 1–2 weeks. Cells should be grownfor at least 3 weeks under selection pressure to avoidcontamination <strong>with</strong> non-resistant cells. Negative controlwells (e.g., sample <strong>with</strong>out expression plasmid) shouldbe inspected by light microscopy and should notcontain any signs of cell growth. Dependent on cell typeand cell growth, selection can be extended up to 4–5weeks or longer in total.■■Analysis of Stable ClonesOnce you have identified resistant clones, expand thecells and assay for your gene of interest by using anappropriate analysis method (e.g., microscopy, flowcytometry, ELISA). For the analysis of the positivecontrol cells, use fluorescence microscopy to screenthe 96-well plate.Technical Information / Transfection9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com425
Guideline for Generation of Stable Cell LinesContinuedTechnical Information / TransfectionTable 2: TroubleshootingSymptomTransient transfection efficiency is lowViability is low 24 hours post-transfectionTransfected cells do not grow in 96-well plates, even<strong>with</strong>out G418Number of resistant clones in 96-well plates is low afterselectionSuggestionOptimal cell density should be determined for each cell type. For adherent cells, the optimalconfluency at the time of transfection is normally 60–80%. Higher, as well as substantiallylower, cell densities may cause lower transfection efficiencies. Suspension cells must be in theirlogarithmic growth phase.Choose appropriate transfection method (e.g., Nucleofection for difficult-to-transfect cell lines).Try lower DNA amounts when using cells known to be DNA sensitive. Check passage number,split rhythm and medium in cell supplier instructions for your cell type. Choose appropriatetransfection method (e.g., Nucleofection for difficult-to-transfect cell lines).Re-titrate plating densitiy for optimal cell growth. Don’t go below minimal cell number for singlecell growth even <strong>with</strong>out selection.Check passage number, split rhythm and medium in cell supplier instructions for your cell type.Use flat-bottomed plates for adherent cells and round-bottomed plates for suspension cells.Check transient transfection efficiency of your transfection method (e.g., using maxGFP ReporterProtein as a control).Try higher DNA amounts.Re-evaluate G418 amount for optimal cell growth in single cell cultures. Try lower G418concentration.Re-check the optimal plating density in 96-well plates. If correct, increase cell numbers per well.Control passage number of cells and confluency of cells before transfection.Choose appropriate transfection method (e.g., Nucleofection for difficult-to-transfect cell lines).After selection, too many resistant clones mixed <strong>with</strong> nonresistantcell clones in 96-well plates Clones are growingon negative control plateClone does not grow out after selectionClone is resistant but gene of interest does not showexpression in several clones checkedPositive control provides high number of resistant clones,but gene of interest does notFeed cells <strong>with</strong> fresh G418 selection medium at least 14 days after transfection.Use the same batch of G418 you used for initial G418 titration.Re-check the optimal plating density in 96-well plates. If correct, decrease cell numbers per well.Use a similar passage number (difference not more than 10) of cells for titration of G418 and fortransfection and selection.Wait 4–5 weeks before picking a resistant clone to obtain a sufficient number of cells for cultureexpansion.Use the same batch (and concentration) of G418 you used for initial G418 titration.Check expression of gene of interest in a transient expression assay, if possible.Try linearizing the plasmid before transfection, this prevents disruption of gene of interestduring integration.Check the sequence of gene of interest for ATG and Stop-Codon.Reverify correct insertion of gene of interest and the resistance marker into plasmidby sequencing or restriction digest.Check whether the expressed recombinant protein is toxic to the cells.9References1. Grimm S. (2004). The art and design of genetic screens:mammalian culture cells. Nature Rev Gen, 5: 179-189.2. Hacein-Bey-Abina, S. et al. (2003). LMO2-associated clonal T cellproliferation in two patients after gene therapy for SCID-X1.Science, 302: 415-419.3. Southern, P. J., Berg, P. (1982) Transformation of mammaliancells to antibiotic resistance <strong>with</strong> a bacterial gene under controlof the SV40 early region promoter.4. J Mol Appl Genet, 1: 327-341.5. Wurm, F. M. (2004). Production of recombinant proteintherapeutics in cultivated mammalian cells. Nature Biotechnol,22: 1393-1398.426North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Designing an RNAi Experiment Using NucleofectionThe Nucleofector Technology is well suited for the transfection of siRNA duplexes or shRNA vectors into both primary cells anddifficult-to-transfect cell lines.Choose siRNA■■Select gene target(s)■■Select control siRNA––Negative control siRNA – e.g., Thermo ScientificsiGENOME Non-Targeting Control*––Positive control siRNA – e.g., siGENOME® GAPDHsiRNA*Confirm siRNA Delivery■■Confirm siRNA delivery efficiency using:––Fluorescently-labeled siRNAor––Fluorescent expression plasmid (e.g., pmaxGFPVector)or––pmaxGFP Vector and maxGFP Reporter ProteinsiRNAor––siRNA targeting housekeeping geneChoose Cell Type and Transfection Protocol■■Select cell type(s) to maximize physiologicalrelevance of results■■Find Optimized Nucleofection Protocols atwww.lonza.com/cell-database■■Select transfection controls––Untreated sample (no siRNA and transfection)––Mock-transfection (no siRNA, only transfection)Choose Detection Assay■■Select detection assay(s)––mRNA – branched-DNA, RT-PCR––Protein – ELISA, Western, FACS analysis––Phenotype – viability, apoptosissTechnical Information / TransfectionOptimize Target Knockdown■■Determine optimal siRNA concentration––100 µl reaction ➞ 0.2–200 pmol (2 n–2 µM)––20 µl reaction ➞ 0.04–40 pmol (2 nM–2 µMAdapt Assay Conditions■■Optimize detection assay(s) conditions for specificsystem––Determine optimal cell densities for linear detectionrange––Correlate results from multiple assays■■Optimize Assay Conditions––Perform detection time course or multiple assays––mRNA ➞ 12–72 hours––Protein ➞ 24–96 hours9Confirm Specificity of Silencing Event■■Confirm gene knockdown results <strong>with</strong> different siRNA reagents––If using Thermo Scientific siGENOME SMARTpool siRNA Reagents*, follow-up <strong>with</strong>Thermo Scientific ON-TARGETplus SMARTpool siRNA Reagents* targeting same genes––If using individual siGENOME® siRNA*, use multiple siRNAs targeting same genes■■If possible, perform rescue experiments*Thermo Fisher Scientific, Dharmacon ProductsEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com427
Designing an RNAi Experiment Using NucleofectionContinuedTechnical Information / Transfection9Establish/Verify Nucleofection Conditions <strong>with</strong>pmaxGFP VectorOptimal Nucleofection Conditions for a particular cell typeare identical whether you are transfecting DNA or RNA.We recommend performing a preliminary experiment <strong>with</strong>pmaxGFP Vector (our positive control plasmid, included inevery kit) in order to establish/verify the optimalNucleofector Solution and Program for your cells. Oncethese conditions have been determined, they remain thesame whether you are transfecting DNA or RNA (or bothtogether).Identify Appropriate Experimental ControlsTo make sure that the conclusions drawn from siRNAexperiments are accurate, it is necessary to include theappropriate experimental controls. We recommend includingat least four types of experimental controls in every RNAiexperiment. Parallel testing of multiple controls underseveral conditions can be easily performed using the96-well Shuttle System.■■Positive siRNA ControlThis should be a validated siRNA pool or individual siRNAtargeting a well-characterized housekeeping gene, suchas cyclophilin B (also known as PPIB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or Lamin. A goodpositive control targeting a well-expressed butnon-essential gene is useful for establishingexperimental parameters <strong>with</strong>out affecting cellularviability. It can also be used as a negative control that isnot associated <strong>with</strong> any particular pathway under study(i.e., it fails to generate an observable phenotype in theassay being employed).■■Negative siRNA ControlNegative siRNA controls are bioinformatically designedand validated to have no known target in the cell type ofchoice. These reagents are important for distinguishingsequence specific silencing from sequenceindependenteffects that are associated <strong>with</strong> thedelivery of siRNA into the cell. Such sequenceindependenteffects can include toxicity resulting fromthe process of transfection in conjunction <strong>with</strong> nucleicacid delivery or hypersensitivity to introduction ofdouble stranded RNA. Investigators are encouraged totest multiple candidates in their own experimentalsystems to empirically confirm that the negativecontrols do not result in any observable and unintendedoff-target effects. For that purpose, Thermo FisherScientific offers a comprehensive portfolio of multiplenegative controls, including the ON-TARGETplus®Non-Targeting Controls, which have been confirmed bymicroarray analysis to have little to no off-targetsignature in HeLa cells.■■Untreated Transfection ControlThe untreated control sample is comprised of cells thathave neither been treated <strong>with</strong> siRNA nor subjected tothe transfection process. This control serves as anindicator of baseline cellular activity to which all otherconditions can be compared.■■Mock-treated ControlThe mock-treated control sample is one in which thecells are subjected to the transfection procedure in theabsence of siRNA. In the case of Nucleofection, thecells would be exposed to the Nucleofector Solutionand subjected to the Nucleofection Procedure in theabsence of siRNA. The analysis of mock-treated cellswill indicate whether the transfection process results incytotoxicity or other non-specific effects.Fine-tune Specific siRNA Sequence/ConcentrationsUsing the same Nucleofection Conditions, simplysubstitute siRNA for the pmaxGFP Vector used in thepreliminary experiment. We often include the pmaxGFPVector (either in a separate parallel sample or co-transfectedby Nucleofection <strong>with</strong> the siRNA) as an easy means ofcomparing relative transfection efficiencies betweenexperiments or selecting transfected cells. The transfectionefficiency using DNA is usually substantially lower ascompared to siRNA. If you are interested in usingfluorescently labeled oligonucleotides, please first read ouradditional notes at the end of this article or contact ourScientific Support Team for specific suggestions.■■Selection of an Optimal siRNA SequenceIf you are using siRNA sequences which have not beenpreviously characterized, we recommend investing aconsiderable amount of time in their selection. Themajority of siRNA providers offer an oligonucleotideoptimization service, however, it is still often necessaryto test several gene-specific siRNA oligonucleotides inorder to find one which efficiently downregulates yourtarget gene.428North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Designing an RNAi Experiment Using NucleofectionContinuedTable 1: Using Low siRNA Concentrations <strong>with</strong> NucleofectionsiRNA Concentration Cell Type Targets/Analysis Method Knockdown Reference2 nM (0.2 pmol*) COS-7 (monkey kidney fibroblast) Bruton’s tyrosine kinase / FACS 96% 57 nM (0.7 pmol*) THP-1 (human monocytic leukaemia) Interferon Regulatory Factor (IRF5) / RT-PCR »strongly reduced« 61 nM (0.1 pmol*) HUVEC (human umbilical vein endothelial cells) Interferon Integrins 1 / 3 and>90% 7Akt / Western Blot Migration* Per cuvette (100 µl volume)■■Determination of the Optimal Effective siRNAConcentrationWhen performing siRNA-mediated knockdownexperiments, it is advisable to conduct a dose-response(concentration) analysis to determine the minimumsiRNA concentration necessary for sufficient targetknockdown on mRNA, protein or functional level. ForNucleofection, the optimal siRNA concentration canrange from lower than 2 nM up to 2 μM, depending onmultiple factors such as the cell type and the half-life ofthe mRNA and/or protein of the gene target. Todetermine the optimal concentration for your cell typeand target, we suggest performing an initial titration ofthe siRNA concentration <strong>with</strong>in the range of 2 nM –2 μM(0.2–200 pmol in 100 μl; 0.04–40 pmol in 20 μl).Starting concentrations for a minimum titration wouldbe 30 and 300 nM. If looking at concentrations, thesevalues may seem higher than <strong>with</strong> lipid-based methods,but it is important to remember that Nucleofectionoccurs in a 5x to 25x lower volume (20 μl <strong>with</strong>Nucleocuvette Strips vs. 100 μl <strong>with</strong> 96-well lipofection;100 μl single Nucleocuvette Vessels or aluminumcuvettes vs. 1–2.5 ml <strong>with</strong> 6-well lipofection).The optimal effective siRNA concentration is dependenton the target and the cell type. Indeed, there arenumerous publications in which Nucleofection of
Designing an RNAi Experiment Using NucleofectionContinuedTechnical Information / Transfection9430AmountWeightMolecular weight of a 21 bpsiRNA ds-oligonucleotide:21 × 660 g/mol = 13860 g/mol= 13.86 ng/pmol ≈ 14 ng/pmolConcentration100 μl Nucleocuvette VesselAdditional Notes■■Measuring Transfection Efficiency Using fluorescentlylabeledsiRNAExperiments <strong>with</strong> fluorescently-labeled siRNAs haveshown transfection efficiencies of up to 99% in somecell types. Unless a confocal microscope or FACS isavailable, the use of fluorescently-labeled siRNA forinitial setup experiments is not advisable, as manyfluorescent labels fade quickly following Nucleofection.Likewise, the amount of fluorescently-labeled siRNAneeded in order to adequately visualize fluorescentcells is often much higher than would be optimal forfunctional response, making this both an expensiveand not highly informative experiment. Furthermore,microscopic analysis may lead to false positive resultsas a result of siRNA sticking to the membrane and notactually entering the cell. We suggest including asample transfecting pmaxGFP Vector in parallel to (orin the same sample as) the siRNA in order to provide ageneral estimate of relative transfection efficiency.Nevertheless, take into account that the transfectionefficiency of siRNA molecules is usually much higher. Ifyou wish to use labeled siRNA for your experiments,please contact our Scientific Support Team to help yourexperiments run as smoothly as possible.■■Enriching for Transfected CellsOne method of enriching for transfected cells is toco-transfect siRNA <strong>with</strong> a plasmid expressing afluorescent reporter or surface marker, and then sortingfor cells expressing the reporter. This approach hasbeen used, for example, in Wu et al. (2005) 4 . UsingshRNA-expressing vectors also allows you to useco-expressed fluorescent or antibiotic resistancemarkers to select for transfected cells (see below).However, transfection efficiencies for plasmid DNA aregenerally lower than those for siRNA duplexes.Concentration20 μl Nucleocuvette Vessel1 pmol 14 ng 1 pmol/100 μl = 10 nmol/l = 10 nM 1 pmol/20 μl = 50 nmol/l = 50 nM5 pmol 69 ng 5 pmol/100 μl = 50 nmol/l = 50 nM 5 pmol/20 μl = 250 nmol/l = 250 nM10 pmol 140 ng 10 pmol/100 μl = 100 nmol/l = 100 nM 10 pmol/20 μl = 500 nmol/l = 500 nM20 pmol 277 ng 20 pmol/100 μl = 200 nmol/l = 200 nM 20 pmol/20 μl = 1000 nmol/l = 1 μM50 pmol 690 ng 50 pmol/100 μl = 500 nmol/l = 500 nM 50 pmol/20 μl = 2500 nmol/l = 2.5 μM100 pmol 1.4 μg 100 pmol/100 μl = 1000 nmol/l = 1 μM 100 pmol/20 μl = 5000 nmol/l = 5 μMFor individual calculation, also refer to our website www.lonza.com/sirna-calculator■■Longterm RNAi Effects (siRNA duplexes vs. shRNA-Expressing Vectors)Chemically synthesized siRNA duplexes offer a rapidmeans for determining the siRNA sequences that resultin efficient knockdown of your target gene, but thisdownregulation is transient (generally persisting 2–5days) and may not be sufficient when silencing targets<strong>with</strong> low turnover rates or in other applications where alonger duration of effect is required. Consequently, anumber of different plasmid vectors that express siRNA,or shRNA (short hairpin RNA) are commerciallyavailable. In addition to enabling long-term expressionof siRNA/shRNA, these vectors have the advantagesthat they can be grown, handled and stored as plasmidDNA, co-express fluorescent markers or antibioticresistance genes (facilitating identification oftransfected cells/selection of stably transfected cells)and can be engineered <strong>with</strong> inducible promoters topermit switching the knockdown phenotype on and off(such as pSuperior, OligoEngine). The ability of theNucleofector Technology to transfect DNA into primarycells (and many cell lines which are difficult orimpossible to transfect by other means) makes itpossible to now use these vectors in virtually any celltype. Although do keep in mind that transfectionefficiencies <strong>with</strong> siRNA oligonucleotides are generallyhigher than <strong>with</strong> plasmid DNA.■■Stability of siRNA Duplexes in Nucleofector SolutionsThe Nucleofector Solutions were tested for RNAseactivity. Incubation of RNA in the solutions for two hoursat 37°C did not affect RNA stability.References1. Persengiev SP, et al. RNA. 2004 Jan;10(1):12-82. Ross J, 1995, Microbiol Rev 59:423-503. Editorial (2003) Wither RNAi. Nat Cell Biol. 5 (6), 489-490.4. Wu et al. (2005) MCB 25(22):9741-97525. Lindvall JM et al. (2005) Immunol Rev 203, 200-215.6. Schoenemeyer A et al. (2005) J Biol Chem 280, 17005-17012.7. Short SM et al. (2005) J Cell Biol 168, 643-653.North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Media, Reagents and SeraCell Culture Technical InformationWe offer an extensive line of cell culture media and reagentsbacked by years of experience and innovation. Through ourown internal efforts as well as work <strong>with</strong> exceptionalcollaborators, we are able to provide ongoing technicalsupport in the form of protocols, detailed product information,and troubleshooting tips for our broad range of media andreagents. In this section we address manyAdaptation of Cell Cultures to Serum-free MediumThe conversion of a particular cell or cell line from growth inserum-containing medium to serum-free medium isachieved through the weaning process. However, weaningis not required for all cell types. Rapid conversion of a cellpopulation to serum-free conditions can be achieved bypelleting the cells and resuspending them in the serum-freemedium. While this may be successful for some types ofcells, a gradual conversion is more likely to yield the desiredresult.Weaning is actually a process by which a subpopulation ofcells that can proliferate in the absence of serum is selected.The degree of difficulty in selecting these cells is a functionof the physical and nutritional requirements of the cells andthe complexity of the serum-free formulation. Conversion ofcells to growth in UltraCULTURE Medium can be relativelysimple because it is a complex formula. Other formulationsmay contain reduced amounts of protein (i.e., UltraCHOMedium and UltraDOMA Medium) or be entirely devoid ofproteins and peptides (i.e., UltraDOMA-PF Medium). Inpractice, these formulations require slightly more attentionduring the weaning process. However, the benefits of a lowprotein serum-free growth environment and subsequentreduction in downstream processing procedures more thanoffset the extra time spent in the weaning process.Maintenance of cellular function is an aspect of the weaningprocess that must be monitored. One needs to ensure thatthe subpopulation selected exhibits the same characteristics<strong>with</strong> respect to cellular function as the population that wasculti vated in the presence of serum. These functions arediverse and may include receptor expression, viralsusceptibility, monoclonal antibody production, andof the commonly asked questions relating to cell culturetechniques by providing instructions and tips for adaptingcultures to serum-free medium, cryopreservation andreconstitution, preparing powdered media, and more. OurScientific Support Department is prepared to assist you<strong>with</strong> many other technical questions and concerns relatedto cell culture and media.recombinant gene expression. In many cases, an increasein product yield has been noted when cells are converted toa serum-free environment. However, each investigatorshould monitor the cellular function of interest to theirapplication during the weaning process.We recommend two protocols for the conversion of cellpopulations to a serum-free environment. These protocolsmay be used for mammalian and invertebrate cell types.The first protocol may be used <strong>with</strong> attachment independentcells or cells that are loosely adherent and do not requiretrypsini zation. It involves the gradual dilution of the serumcontainingmedium <strong>with</strong> serum-free medium. The secondprotocol may be used <strong>with</strong> both attachment dependent andindependent cell types and begins <strong>with</strong> the serum-freemedium supplemented <strong>with</strong> serum. A gradual reduction inthe serum concentration is performed at each subcultureuntil serum-free growth is achieved. This latter protocol hasthe added advantage of establishing the limit of serumconcentration for the cell type. Some cells (especiallytransfected lines) require small amounts of serum (i.e.,0.1–0.5% v/v). This method allows the investigator to titratethe serum to the lower limit.The two weaning protocols are presented on the followingpage. They represent our recommended procedures,however, each investigator may choose to makemodifcations that better suit their particular application. Inour experience, the minimum cell density maintainedduring the conversion process has a major effect on theoutcome. We recommend that the cells be maintainedabove 3.0 × 10 5 /ml for attachment independent and above30% confluency for attachment dependent cells.Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com431
Protocols for Weaning Cell CulturesTechnical Information / Media, Reagents and SeraProtocol #1: Medium Replacement – for AdherentIndependent Cells (suspensions)Approximate time required: 2 weeks – 6 weeksCulture conditions:––Mammalian cells: 95% air, 5% CO 2, 35°C–37°C––Invertebrate cells: air, 25°C–27°C1. Begin <strong>with</strong> cultures at maximum cell density.2. NOTE: Attachment dependent cells that are exposed totrypsin during subculturing should be converted toserum-free growth using Protocol #2.3. Split cells 1:2 using serum-free medium as the diluent.4. Incubate cells until the maximum cell density isachieved.5. Split cells 1:5 or to 3.0 × 10 5 cells/ml for attachmentindependent cells or 30% confluency for attachmentdependent cells using serum-free medium as thediluent.6. Incubate cells until the maximum cell density isachieved.7. If the cell viability is >85% at this point, and thegeneration time is similar to that observed <strong>with</strong> serumcontainingmedium, the culture may be maintained inserum-free medium using a similar split schedule asoriginally optimized for serum-containing medium.8. If the cells exhibit slow growth or low viability, maintainthe split ratio at 1:2 or 1:5 for 3 successive splits. Theminimum cell density should be above 3.0 × 10 5 cells/ml or 30% confluency during this period.9. Gradually increase the split ratio to obtain a maximumvalue for the cell type being used.NOTE: Some cells may require a small amount of serum for growth. Ifthe cells have not adapted to serum-free cultivation using the aboveprotocol, add 0.1%–0.5% serum to the culture or contact ScientificSupport at scientific.support.eu@lonza.com.Protocol #2: Serum Dilution – for AdherentDependent CellsApproximate time required: 2 weeks – 6 weeksCulture conditions:––Mammalian cells: 95% air, 5% CO 2, 35°C–37°C––Invertebrate cells: air, 25°C–27°C1. Begin <strong>with</strong> cultures at maximum cell density.2. Trypsinize attachment dependent cultures and transferto an appropriately sized centrifuge tube. Attachmentindependent cells may be transferred directly to thecentrifuge tube.3. Sediment the cells by centrifugation at 350 × g for5 minutes.4. Resuspend the cells in serum-free medium containing5% serum (v/v).5. Adjust the cell concentration using the serumsupplemented serum-free medium to a maximum of3.0 × 105 cells/ml for attachment independent cells or adensity to achieve not less than 30% confluency forattachment dependent cells.6. Plant the cells and incubate until a maximum celldensity is achieved.7. Repeat steps 2–6 using a lower concentration of serumat each split. We recommend beginning at 5% serumand lowering to 2%, 1%, 0.5%, and finally 0.1% prior toeliminating serum from the culture.NOTE: If the culture viability drops below 80% or if the generation timeincreases markedly following a decrease in the serum concentration,increase the serum level to the previous value and maintain the cellsfor 2 split cycles before lowering the level of serum again. It may benecessary to institute a more gradual decline in serum concentration<strong>with</strong> these cells. Some cell types may require a small amount of serumfor growth. If the cells have not adapted to serum-free cultivation usingthe protocol described above, add 0.1–0.5% serum to the culture orcontact Scientific Support at scientific.support.eu@lonza.com.9432North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cryopreservation and ReconstitutionBasic Procedure for Cryopreservation andReconstitution of Cultured CellsNOTE: Not applicable to Clonetics and Poietics Primary Humanor Animal Cells.Cryopreservation1. Select a flask of cells at or near confluency.2. Cells should be first removed by trypsinization.3. Adjust the cell concentration to between 2 × 10 6 and8 × 10 6 cells/ml <strong>with</strong> EMEM (Cat. No. 12-136) containing20% Fetal Bovine Serum (Cat. No. 14-501). Centrifugationmay be necessary.4. Add the above cell suspension to an equal volume ofcold ( + 4°C) Cryoprotective Freezing Medium(Cat. No.12-132A).5. Mix continuously to ensure homogeneity.6. Dispense either 4 ml of the cells suspended inCryoprotective Freezing Medium into 5 ml glassampoules, or 1 ml into plastic screw cap vials suitablefor freezing in the liquid or vapor phase of liquid nitrogen.7. Cells are now ready for the freezing cycle. Cells shouldnot be allowed to remain in the Cryoprotective FreezingMedium for more than 1 hour before freezing.8. The temperature of the contents in the ampoule mustthen be lowered at a rate of 0.5°C–2°C/minutethroughout the range of +4°C to -30°C.9. After the temperature has reached -30°C, the rate of thetemperature drop to -70°C (which is the warmesttemperature at which cells can be stored) can be donevery quickly. An automatic programmable freezersystem is the most reliable means of obtainingcontrolled rate freezing.10. Storage of the ampoules or vials must be at -70°C orcolder. A storage temperature of -196°C (liquid nitrogen)is best. It is essential that the temperature of thecontents of the ampoule be -70°C or colder at all timesuntil reconstitution. For prolonged or indefinite storage,the use of liquid nitrogen is strongly recommended.Storage in dry ice or a mechanical freezer (-70°C)should be limited to less than3 months.Ampoule Handling RecommendationsReceipt of AmpouleUpon receipt, transfer the ampoule(s) from the shippingcontainer to a -70°C freezer or a vapor phase nitrogen tank.For long-term storage (over 3 months), a vapor phasenitrogen tank is preferable to prevent significant loss ofviability. Immersion of screw cap ampoules in liquid nitrogenis not recommended.To UseCaution: Wear protective facemask and clothing asampoule explosions can occur.1. Remove an ampoule from the freezer and place it intoa 37°C waterbath. Do not submerge the ampoule orallow water to get under the cap.2. After thawing, disinfect the ampoule <strong>with</strong> 70%isopropanol, then open aseptically in a laminar flowsafety cabinet. Dilute the contents 1:10 in theappropriate growth medium.3. Determine the viability and cell concentration of thethawed cells by using the Trypan Blue exclusion cellcounting method.4. Adjust the cell concentration as desired for seedingculture vessels.5. Twenty-four hours after cells have been seeded, removethe medium and re-feed <strong>with</strong> the appropriate growthmedium. This will remove the cryopreservative, if thealternative method described below is not used.6. As an alternative, the cryopreservative may be removedprior to cell viability determination by centrifugation.This is done by centrifuging the resuspended cells atlow speed for 10 minutes(200 × g). The supernatant isremoved and the cells are resuspended in theappropriate growth medium.ReferenceFreshney, R.I. (2000) Culture of Animal Cells: A Manual of BasicTechnique, 4th edition, Wiley-Liss, Inc., New York, pp. 297–308.Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com433
Determination of Cell NumbersTechnical Information / Media, Reagents and Sera9Counting cells by use of a hemacytometer is a convenientand practical method of determining cell numbers insuspension culture or from dispersed monolayer cultures.The hemacyto meter consists of two chambers, each ofwhich is divided into nine 1.0 mm squares. A cover glass issupported 0.1 mm over these squares so that the totalvolume over each square is 1.0 mm × 0.1 mm or 0.1 mm3,or 10 –4 cm3. Since 1 cm3 is approximately equivalent to 1ml, the cell concentration per ml will be the average countper square × 10 4 .■■Hemacytometer Counts are Subject to the FollowingSources of Error:––Unequal cell distribution in the sample.––Improper filling of chambers.––Failure to adopt a convention for counting cells incontact <strong>with</strong> boundary lines or <strong>with</strong> each other.––Statistical error.With careful attention to detail, the overall error can bereduced to about 15%. It is assumed that the total volume inthe chamber represents a random sample. This will not be avalid assumption unless the suspension consists ofindividual separated cells. Cell distribution in thehemacytometer chamber depends on the particle number,not particle mass. Thus, cell clumps will distribute the sameas single cells and can distort the final result. Unless 90% ormore of the cells are free from contact <strong>with</strong> other cells, thecount should be repeated <strong>with</strong> a new sample. Cells that aredifficult to obtain in uniform suspensions, or in whichextensive clumping cannot be avoided, may be counted byseparating nuclei. This method is more time-consumingthan direct counting and is subject to additional error if thepopulation contains multinucleate cells. A sample will notbe representative if the cells are permitted to settle beforea sample is taken. Always mix the cell suspensionthoroughly before sampling.With a 10X objective and a 10X ocular, one square (1 mm 2 )will approximately fill the microscope field (the circle on therepresentation of a hemacytometer grid). The cellsuspension should be diluted so that each such square hasbetween 20 and 50 cells (2–5 × 10 5 cells/ml). A total of 300to 400 cells should be counted since the counting error isapproximated by the square root of the total count. Acommon convention is to count cells that touch the middleline (of the triple lines) to the left and top of the square, butnot to count cells similarly located to the right and bottom(see diagram).= count; = do not countDiagram of a hemacytometer, improved Neubauer ruling, 0.1 mm deepBrackets indicate 1 mm 2 squares. Circle is the approximate area coveredat 100X magnification.In order to fill the hemacytometer chamber properly bycapillary action, the cover slip, chamber, and the pipetteused to fill the chamber must be scrupulously clean. Thechamber and cover slip are cleaned first <strong>with</strong> distilled water,then <strong>with</strong> absolute ethanol, and wiped dry.Hemacytometer counts do not distinguish between livingand dead cells. A number of stains are useful to makethis distinction. Trypan Blue, among others (erythrosin B,nigrosin), is excluded by the membrane of the viable cells,whereas the nuclei of damaged or dead cells take up thestain. Although this distinction has been questioned, it hasthe virtue of being simple and giving a good approximation.If more than 20% of the cells are stained, the result isprobably significant.434North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Determination of Cell NumbersContinuedMaterials1. Clean hemacytometer and glass coverslip2. Pasteur pipettes3. Hanks’ Balanced Salt Solution (HBSS)(Cat. No. 10-543)4. Trypan Blue, 0.4% in BSS (Cat. No. 17-942E)5. Microscope6. Tubes (12 × 75 mm)7. Hand counter8. Cell suspensionProcedure1. Dilute 0.2 ml of Trypan Blue <strong>with</strong> 0.8 ml of HBSS.2. Place glass coverslip over hemacytometer chamber.3. Transfer 0.5 ml of agitated cell suspension to a 12 × 75mm tube and add 0.5 ml of diluted Trypan Blue.4. With Pasteur pipette, fill both chambers of thehemacytometer (<strong>with</strong>out overflow) by capillary action.Cells will settle in the tube and in the pipette by gravity<strong>with</strong>in a few seconds. Work quickly.5. Using a microscope <strong>with</strong> a 10X ocular and a 10Xobjective, count the cells in each of 10 squares (1 mm2each). If over 10% of the cells represent clumps, repeatentire sequence. If fewer than 200 or more than 500cells are present in the 10 squares, repeat <strong>with</strong> a moresuitable dilution factor.6. Calculate the number of cells per ml, and total numberof cells in the original culture as follows:Cells/ml = average count per square × 10 4 × dilution factor(i.e., 2, if0.5 ml of cells plus 0.5 ml of Trypan Blue is used)Total cells = cells/ml × total volume of cell preparation from whichsample was taken7. Repeat count to check reproducibility.Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com435
Powdered Media PreparationTechnical Information / Media, Reagents and SeraPreparation of Media for FiltrationPowdered media or salt mixtures are extremely hygroscopicand must be protected from atmospheric moisture. Theentire contents of each package should be used immediatelyafter opening. The preparation of medium in concentratedform is not recommended as some free-base amino acidshave low solubility coefficients and insoluble salt complexesmay precipitate in concentrated solution. All powderedproducts do not contain sodium bicarbonate, <strong>with</strong> theexception of UltraCHO Medium (Cat. No. 15-724).Supplements can be added prior to filtration or introducedaseptically to sterile medium.NOTE: The nature of the supplement may affect the storage conditions andshelf life of the medium.Procedure1. Select a suitable container as close in size to the finalvolume as possible. Measure out 90% of the final volumeof deionized or distilled water (cell culture grade wateris available from Lonza in volumes from 500 ml to200 l).2. While gently stirring, add the powdered medium or saltmixture. Continue stirring until dissolved. Do not heatthe water.3. Rinse the original package <strong>with</strong> a small amount ofdeionized or distilled water. Add to solution.4. For each liter of final volume of medium being prepared,add to the solution the required amount of sodiumbicarbonate and/or L-glutamine (consult tables below).Continue stirring until completely dissolved.5. While stirring, adjust the pH to 0.2–0.3 pH units belowthe desired pH. Use 1 N HCl or 1 N NaOH. The pH willnormally rise 0.1–0.3 units during filtration.6. Add additional deionized or distilled water to bring themedium to final volume.7. Sterilize immediately by filtration using a 0.22-micronor smaller filter. To reduce the loss of CO 2, positivepressure (3–15 psi), <strong>with</strong> an inert gas (i.e., nitrogen)should be used for filtering. The use of CO 2is notrecommended, as it will alter the pH of the medium.8. After filtration, aseptically transfer into sterilecontainers. Store medium at 2°C–4°C in the dark untilready to use.9Sodium Bicarbonate Addition TableProduct Description Cat. No. NaHCO 3Solution ml/lNaHCO 3Powder g/l17-613 15-613DMEM w/L-glutamine 15-604 49.3 3.700DMEM w/o L-glutamine 15-614 49.3 3.700MEM Eagle w/L-glutamine 15-611 29.3 2.200RPMI w/L-glutamine 15-702 26.7 2.000L-glutamine Addition TableProduct Description Cat. No. L-glutamineSolution ml/lDMEM w/o L-glutamine 15-614 20.0(4.0 mM)L-glutaminePowder g/L17-605 15-6050.585(4.0 mM)436North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Subculturing Procedures for Mammalian CellsNOTE: Not applicable to Clonetics and Poietics Primary Human or AnimalCells.In cell culture there is frequently the need to subculturecells. In doing so, cells can be propagated for the purposesof increasing cell numbers or providing cells in a culturevessel suitable to one’s needs. There are a number of waysto remove cells from one culture vessel and pass them toanother vessel. Cells may be removed from surfaces onwhich they are attached by:––Mechanical means (scraping)––Chelating agents, ethylenediaminetetraacetic acid(EDTA)––Enzymes (trypsin, pronase, collagenase)Enzymes and chelating agents are often used in combi nation.Trypsin is an aqueous crude extract prepared from porcinepancreas. It is the most common means used for removal ofcells from surfaces and from intact tissue. Trypsin is, tosome extent, a misnomer because in addition to trypsin,the preparation contains other proteases, lipases, andcarbohydrases. The multitude of digestive enzymesproduced by the pancreas would be expected to be found intrypsin preparations. Pure crystalline trypsin can be used,but it is more expensive than crude trypsin and often doesnot work as well, especially when preparing cells from intacttissue.The optimum conditions for trypsin activity are a pH rangeof 7.6–7.8 and a temperature of 37°C. The effect of trypsin isto break down the intracellular matrix that binds cells toeach other or to a substrate surface.There are no chemical standards for trypsin activity. Weconduct quality assurance tests on trypsin to determine itscapacity to detach cells from a substrate surface in astandard time period <strong>with</strong>out damage. This is in addition tothe usual tests for sterility.Trypsin is typically used at concentrations between 0.05%and 0.25%, although some applications may requireconcentrations outside this range. Versene® (EDTA)enhances trypsin action, and therefore lowers the requiredtrypsin concentration for effective performance.Concentrated trypsin (2.5%, Cat. No. 17-160) should bediluted in calcium- and magnesium-free balanced saltsolution (BSS) (Hanks’ BSS, Cat. No. 10-543; or Dulbecco’sPhosphate Buffered Saline, Cat. No. 17-512). Dilution inwater is not recommended since the solution will behypotonic and produce cell damage. Dilution in saline aloneis also damaging to cells.Trypsinization ProcedureCell cultures are normally subcultured (“split”) when thecultures are at or near confluency. As a general rule, thelonger the time frame between when confluency is firstachieved and subculturing, the longer it will take for thetrypsin to act.1. Decant medium from the culture vessel. Serum inhibitstrypsin activity, so complete removal of serumcontainingmedium is necessary.2. Rinse the cell sheet <strong>with</strong> BSS <strong>with</strong>out calcium andmagnesium before addition of Trypsin/Versene®(Cat. No. 17-161). The monolayer should be thoroughlycovered <strong>with</strong> BSS. This rinse is instantaneous but theBSS can remain on the cell sheet for up to 4 hours, ifdesired.3. Pour off rinse medium. Trypsin/Versene® is to be addedto each vessel as follows:75 cm 2 flask 2.5 ml to 5.0 ml150 cm 2 flask 5.0 ml to 10.0 ml850 cm 2 roller bottle 10.0 ml to 20.0 ml4. Cover the monolayer thoroughly <strong>with</strong> Trypsin/Versene®.Since different lots of Trypsin/Versene® may vary instrength, it is acceptable to monitor the trypsinizationprocess at room temperature for the first 30 seconds.This will ensure that the trypsinization process is notoccurring too rapidly.5. The culture vessel should then be moderately hitagainst the palm of the hand to see if the cells are beingdislodged. Hold the vessel up to a light in a verticalposition and look for signs of the cell sheet sloughingoff of the surface. If the entire monolayer is dislodged,proceed to step #6. If not, incubate at 37°C and observethe vessel every minute for dissociation. The culturevessel should again be hit against the palm of the handto ensure all cells have been dislodged. Remove culturevessel from the incubator.Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com437
Subculturing Procedures for Mammalian CellsContinuedTechnical Information / Media, Reagents and Sera96. Immediately transfer dissociated cells to a vesselcontaining medium supplemented <strong>with</strong> 10% serum. Allof the cells should be removed. Aspirate the mediumplus cells <strong>with</strong> a pipette onto the surface to remove allremaining cells. It is essential that this aspiration bedone as completely as possible <strong>with</strong> a small borepipette so as to obtain individual, dispersed cells. If thecells are not separated, the new culture will containnumerous microcolonies. Cells added to the vesselshould be stirred <strong>with</strong> a magnetic stir bar at a speedthat avoids vortexing (approximately 100–200 rpm), oragitated frequently. It is important at this point to addmedium containing serum at least 10 times the volumeof Trypsin/Versene® used. This will ensure that thedigestive agent is inhibited.7. Add sufficient fresh medium to the aspirated suspensionso that the total volume will cover the surface of twoculture vessels, each having the same surface area asthe original culture vessel (or use a single culturevessel having twice the floor area of the original vessel).This is a 1:2 split. Other split ratios can be used forvigorously growing cell populations.8. Incubate the culture vessel (or vessels) at 37°C.9. When making 1:2 splits, subculturing of human diploidcell cultures should be done on a rigid 3 or 4 dayschedule, at which time confluent sheets should occur.Surplus cells can be frozen and stored in liquid nitrogen.10. Populations that can be cultivated indefinitely can besubcultured serially each time confluency is reached. Ifthe culture is a diploid population <strong>with</strong> a finite doublingcapacity, increase the population doubling level (PDL)number by one at each 1:2 subculturing (split).11. By making repeated 1:2 splits (twice a week) it can beseen that the number of culture vessels can be built upgeo metrically (1, 2, 4, 8, 16, 32, 64, etc.) in a shortperiod of time for the production of large quantities ofcells for various purposes.12. Although the line will be eventually lost as a continuouslypassaged line, it will not be lost for use since frozenampoules can be obtained at almost every passage andthus the line can be restored to continuous passageagain, up to a cumulative total of about 50 populationdoublings. By repeating this procedure, the number ofcells that can be obtained is almost unlimited for allpractical purposes.13. A human embryonic diploid line has an in vitro life spanof about fifty 1:2 subcultivations, or populationdoublings, at which time the cells will cease to divideand eventually die.14. Using split ratios higher than 1:2 results in theadvantage of minimizing the number of manipulationsnecessary to obtain a specific cell density or number ofculture vessels. Since human embryonic diploid celllines pass through a finite number of populationdoublings in vitro, it is necessary to keep a record of thenumber of population doublings that have elapsed. Witha 1:2 split ratio this is achieved by simply adding “1” toeach split since this ratio yields one population doubling.Larger split ratios can be used. For example, a split ratioof 1:4 would yield 2 doublings per 1:4 split; a 1:8 splitratio would yield 3 doublings per 1:8 split. In order tohave knowledge of the approach of cessation, it isessential to keep records of the number of elapsedpopulation doublings.15. Since human diploid cells multiply by fission, theincrease in population may be expressed per cell asfollows:1 2 4 8 16 …Number of cells0 1 2 3 4 …Population Doubling LevelReferences1. Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation ofhuman diploid cell strains. Exp. Cell <strong>Research</strong> 25:585.2. Hayflick, L. (1970) Aging under glass. Exp. Geront. 5:291.3. Hayflick, L. (1965) The limited in vitro lifetime of human diploidcell strains. Exp. Cell Res. 37:614.4. Hayflick, L. (1968) Human cells and aging. Scientific American218:32.5. Hayflick, L. (1973) Subculturing human diploid fibroblastcultures. Methods and Applications of Tissue Culture Eds.Patterson, M.K. and Kruse, P.F., Academic Press, N.Y.6. Freshney, R.I. (1983) Culture of Animal Cells: A Manual of BasicTechnique. Alan R. Liss, Inc., New York.438North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaBasal Medium Eagle (BME)Cat. No.Component12-132 (Freezing medium)Liquid mg/l12-105Liquid mg/lInorganic SaltsCaCl 2•2H 20 158.00 265.00KCl 340.00 400.00KH 2PO 451.00MgSO 4•7H 2O 170.00 200.00NaCl 6,800.00 6,800.00NaHC0 3298.00 2,200.00Na 2HPO 4•7H 2O 77.00NaH 2PO 4(anhyd.) 140.00Other ComponentsDimethylsulfoxide 150 mlGlucose 850.00 1,000.00Phenol Red•Na 17.00 10.00Amino AcidsL- Arginine•HCl 17.94 21.10L -Cystine 10.20 12.00L -Histidine•HCl•H 20 8.93 10.50L -Isoleucine 22.27 26.20L -Leucine 22.27 26.20L -Lysine•HCl 31.03 36.50L -Methionine 6.38 7.50L -Phenylalanine 14.03 16.50L -Threonine 20.23 23.80L -Tryptophan 3.40 4.00L -Tyrosine 15.39 18.10L -Valine 19.89 23.40Vitaminsd-Biotin 0.21 0.24D-Ca Pantothenate 0.20 0.24Choline Chloride 0.12 0.14Folic Acid 0.38 0.44Nicotinamide 0.10 0.12Pyridoxine•HCl 0.17 0.20Riboflavin 0.03 0.04Thiamine•HCl 0.29 0.34Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com439
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and Sera9Dulbecco’s Modified Eagle’s Medium (DMEM)Cat. No.Component12-604Liquidmg/l15-604Powdermg/l12-614Liquidmg/l15-614Powdermg/l12-707Liquidmg/l12-708Liquidmg/l12-709Liquidmg/l12-733Liquidmg/l12-741Liquidmg/l12-914Liquidmg/l12-917Liquidmg/lBE12-6 0 /4U1Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00Fe(NO 3) 3•9H 20 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00MgSO 4(anhyd.) 97.66 97.66MgSO 4•7H 20 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00NaCl 6,400.00 6,400.00 6,400.00 6,400.00 6,400.00 5,400.00 5,400.00 6,400.00 6,400.00 6,400.00 6,400.00 6,400.00NaHC0 33,700.00 3,700.00 3,700.00 3,700.00 3,700.00 3,700.00 3,700.00 3,700.00 3,700.00 3,700.00NaH 2PO 4108.69 108.69NaH 2P0 4•H 20 125.00 125.00 125.00 125.00 125.00 125.00 125.00 125.00 125.00 125.00Other ComponentsGlucose 4,500.00 4,500.00 4,500.00 4,500.00 1,000.00 1,000.00 4,500.00 4,500.00 4,500.00 4,500.00 4,500.00 4,500.00HEPES 5,957.50 5,957.50Phenol Red 15.00 15.00 15.34 15.00 15.34 15.00 15.00 15.00 15.00 15.00 15.00Sodium Pyruvate 110.00 110.00 110.00 110.00 110.00 110.00 110.00 110.00Amino AcidsL-Arginine•HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00L-Cystine 48.00 48.00 48.00 48.00 48.00 48.00 48.00 48.00 48.00 48.00L-Cystine•2HCl 62.58 62.58L-Glutamine 584.00 584.00 584.00L-Alanyl-L-Glutamine868.00(UltraGlutamine I)Glycine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00L-Histidine•HCl•H 20 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00L-Isoleucine 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80L-Leucine 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80 104.80L-Lysine•HCl 146.20 146.20 146.20 146.20 146.20 146.20 146.20 146.20 146.20 146.20 146.20 146.20L-Methionine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00L-Serine 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00L-Threonine 95.20 95.20 95.20 95.20 95.20 95.20 95.20 95.20 95.20 95.20 95.20 95.20L-Tryptophan 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00L-Tyrosine 72.00 72.00 72.00 72.00 72.00 72.00 72.00 72.00 72.00 72.00L-Tyrosine•2Na 103.79 103.79L-Valine 93.60 93.60 93.60 93.60 93.60 93.60 93.60 93.60 93.60 93.60 93.60 93.60VitaminsD-Ca Pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Choline Chloride 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Folic Acid 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00i-Inositol 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.00Nicotinamide 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Pyridoxine•HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Riboflavin 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40Thiamine•HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00Dulbecco, R. and G. Freeman (1959) Virology 8:396–397.440North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedDMEM: F-12Cat. No.Component12-719Liquid mg/lBE04-687Liquid mg/lBE04-68 7 /U1Liquid mg/lInorganic SaltsCaCl 2(anhyd.) 116.00 116.00 116.00CuSO 4•5H 20 0.0012 0.0012 0.0012Fe(NO 3) 3•9H 20 0.05 0.05 0.05FeSO 4•7H 20 0.42 0.42 0.42KCl 311.83 311.83 311.83MgCl 2•6H 20 61.00 61.00 61.00MgSO 4•7H 20 100.00 100.00 100.00NaCl 6,999.50 6,999.50 6,999.50NaHCO 31,200.00 1,200.00 1,200.00Na 2H 2P0 4•7H 20 134.00 134.00 134.00NaH 2P0 4•H 20 62.50 62.50 62.50ZnSO 4•7H 20 0.43 0.43 0.43Other ComponentsGlucose 3,151.00 3,151.00 3,151.00HEPES 3,574.50Hypoxanthine 2.04 2.04 2.04Linoleic Acid 0.04 0.04 0.04Lipoic Acid 0.10 0.103 0.103Phenol Red•Na 8.00 8.00 8.00Putrescine•2HCl 0.08 0.08 0.08Sodium Pyruvate 110.00 110.00 110.00Thymidine 0.36 0.36 0.36Amino AcidsL-Alanine 4.46 4.46 4.46L-Arginine•HCl 147.35 147.35 147.35L-Asparagine•H 20 7.50 7.50 7.50L-Aspartic Acid 6.66 6.66 6.66L-Cysteine•HCl•H 20 17.56 17.56 17.56L-Cystine 24.00 24.00 24.00L-Glutamic Acid 7.36 7.36 7.36L-Glutamine 365.10 365.10L-Alanyl-L-Glutamine868.00(UltraGlutamine 1)Glycine 18.76 18.76 18.76L-Histidine•HCl•H 20 31.48 31.48 31.48L-Isoleucine 54.37 54.37 54.37L-Leucine 58.96 58.96 58.96L-Lysine•HCl 91.37 91.37 91.37L-Methionine 17.24 17.24 17.24L-Phenylalanine 35.48 35.48 35.48L-Proline 17.27 17.27 17.27L-Serine 26.26 26.26 26.26L-Threonine 53.56 53.56 53.56L-Tryptophan 9.02 9.02 9.02L-Tyrosine 38.72 38.72 38.72L-Valine 52.66 52.66 52.66DMEM: F-12Cat. No.ComponentHam’s Medium (F-12 and F-10)Cat. No.Component12-719Liquid mg/lHam’s F-1212-615Liquid mg/lBE04-687Liquid mg/lHam’s F-1012-618Liquid mg/lBE04-68 7 /U1Liquid mg/lVitaminsd-Biotin 0.004 0.004 0.004D-Ca Pantothenate 2.12 2.12 2.12Choline Chloride 8.98 8.98 8.98Folic Acid 2.66 2.66 2.66i-Inositol 12.51 12.50 12.50Nicotinamide 2.02 2.02 2.02Pyridoxine•HCl 2.03 2.03 2.03Riboflavin 0.22 0.22 0.22Thiamine•HCl 2.17 2.17 2.17Vitamin B 120.68 0.68 0.68Europe onlyHam’s F-10BE02-014Liquid mg/lInorganic SaltsCaCl 2•2H 20 44.11 44.10 44.10CuSO 4•5H 20 0.0025 0.0025 0.0025FeSO 4•7H 20 0.83 0.83 0.83KCl 223.65 285.00 285.00KH2PO 483.00 83.00MgCl 2•6H 20 122.00MgSO 4•7H 20 152.80 152.80NaCl 7,599.00 7,400.00 7,400.00NaHCO 31,176.00 1,200.00 1,200.00Na 2HPO 4•7H 20 268.10 290.00 290.00ZnSO4•7H20 0.86 0.29 0.29Other ComponentsGlucose 1,801.60 1,100.00 1,100.00Hypoxanthine 4.08 4.08 4.08Linoleic Acid 0.08Lipoic Acid 0.21 0.20 0.20Phenol Red•Na 1.24 1.20 1.20Putrescine•2HCl 0.16Sodium Pyruvate 110.00 110.00 110.00Thymidine 0.73 0.73Amino AcidsL-Alanine 8.91 8.91 8.91L-Arginine•HCl 210.70 211.00 211.00L-Asparagine•H 20 15.01 15.00 15.00L-Aspartic Acid 13.31 13.30 13.30L-Cysteine•HCl•H 20 35.12 35.13 35.13L-Glutamic Acid 14.71 14.70 14.70L-Glutamine 146.20 146.20L-Alanyl•L-Glutamine 446.00Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com441
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and Sera9Ham’s Medium (F-12 and F-10)Cat. No.Component1. Ham, R.G. (1965) Proc. Natl. Acad. Sci. USA 53: 288–293.2. Ham, R.G. (1963) Exp. Cell Res. 29:515 – 526.Grace’s Insect MediumCat. No. 04-457 04-649 04-501Component Liquid mg/l Liquid mg/l Liquid mg/lInorganic SaltsHam’s F-1212-615Liquid mg/lCaCl 2•2H 2O 993.00 993.00 993.00KCl 4,100.00 4,100.00 4,100.00MgCl 2•6H 2O 2,280.00 2,280.00 2,280.00MgSO 4(anhyd.) 1,358.00Ham’s F-1012-618Liquid mg/lEurope onlyHam’s F-10BE02-014Liquid mg/lGlycine 7.51 7.51 7.51L-Histidine•HCl•H 20 20.96 21.00 23.00L-Isoleucine 3.94 2.60 2.60L-Leucine 13.12 13.10 13.10L-Lysine•HCl 36.54 29.30 29.30L-Methionine 4.48 4.48 4.48L-Phenylalanine 4.96 4.96 4.96L-Proline 34.53 11.50 11.50L-Serine 10.51 10.50 10.50L-Threonine 11.91 3.57 3.57L-Tryptophan 2.04 0.60 0.60L-Tyrosine 5.44 1.81 1.81L-Valine 11.71 3.50 3.50Vitaminsd-Biotin 0.007 0.024 0.024D-Ca Pantothenate 0.24 0.72 0.72Choline Chloride 13.96 0.70 0.70Folic Acid 1.32 1.32 1.32i-Inositol 18.00 0.54 0.54Nicotinamide 0.037 0.620 0.62Pyridoxine•HCl 0.062 0.210 0.21Riboflavin 0.038 0.380 0.38Thiamine•HCl 0.34 1.01 1.01Vitamin B 121.36 1.36 1.36MgSO 4•7H 2O 2,780.00 2,780.00NaHCO 3350.00 350.00 350.00NaH2PO4•H2O 1,008.00 1,008.00 1,008.00Other ComponentsFetal Bovine Serum10%(v/v)(HeatInactivated)Fructose 400.00 400.00 400.00Fumaric Acid 55.00 55.00 55.00Gentamicin Sulfate 50.00 55.00Grace’s Insect MediumCat. No. 04-457 04-649 04-501Component Liquid mg/l Liquid mg/l Liquid mg/lGlucose 700.00 700.00 700.00α-Ketoglutaric Acid 370.00 370.00 370.00Lactalbumin Hydrolysate 3,330.00 3,330.00Malic Acid 670.00 670.00 670.00Succinic Acid 60.00 60.00 60.00Sucrose 26,680.00 26,680.00 26,680.00Yeastolate 3,330.00 3,330.00Amino AcidsL-Alanine 225.00 225.00 225.00ß-Alanine 200.00 200.00 200.00L-Arginine•HCl 700.00 700.00 700.00L-Asparagine•H 2O 398.00 398.00 398.00L-Aspartic Acid 350.00 350.00 350.00L-Cystine•2HCl 28.29 28.29 28.29L-Glutamic Acid 600.00 600.00 600.00L-Glutamine 600.00 600.00 600.00Glycine 650.00 650.00 650.00L-Histidine 2,500.00 2,500.00 2,500.00L-Isoleucine 50.00 50.00 50.00L-Leucine 75.00 75.00 75.00L-Lysine•HCl 625.00 625.00 625.00L-Methionine 50.00 50.00 50.00L-Phenylalanine 150.00 150.00 150.00L-Proline 350.00 350.00 350.00DL-Serine 1,100.00 1,100.00 1,100.00L-Threonine 175.00 175.00 175.00L-Tryptophan 100.00 100.00 100.00L-Tyrosine•2Na•2H 2O 72.08 72.08 72.08L-Valine 100.00 100.00 100.00Vitaminsp-Aminobenzoic Acid 0.02 0.02 0.02d-Biotin 0.01 0.01 0.01D-Ca Pantothenate 0.02 0.02 0.02Choline Chloride 0.20 0.20 0.20Folic Acid 0.02 0.02 0.02i-Inositol 0.02 0.02 0.02Nicotinic Acid 0.02 0.02 0.02Pyridoxine•HCl 0.02 0.02 0.02Riboflavin 0.02 0.02 0.02Thiamine•HCl 0.02 0.02 0.02442North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedInsect MediumCat. No.ComponentSchneider’s04-351Liquid mg/lEurope onlyTC-100BE02-011Liquid mg/lInorganic SaltsCaCl 2(anhyd.) 600.00 996.66KCl 1,600.00 2,870.00KH 2PO 4450.00MgCl 2(anhyd.) 1,068.19MgCl 2•6H 2OMgSO 4(anhyd.) 1,357.86MgSO 4•7H 2O 3,700.00MnCl 2•4H 2O 1,321.00NaCl 2,100.00NaHCO 3400.00 350.00NaH2PO4•H2O 876.92Other ComponentsFumaric Acid 100.00Glucose 2,000.00 1,000.00α-Ketoglutaric Acid 200.00L-Malic Acid 100.00Succinic Acid 100.00Trehalose•2H 2O 2,000.00Tryptose Broth 2,600.00Yeastolate 2,000.00Amino AcidsL-Alanine 225.00ß-Alanine 500.00 700.00L-Arginine•HCl 350.00L-Arginine•FB 400.00L-Asparagine 1,300.00 350.00L-Aspartic Acid 400.00 350.00L-Cysteine 60.00L-Cystine 100.00 19.20L-Glutamic Acid 800.00 600.00L-Glutamine 1,800.00 600.00Glycine 250.00 650.00L-Histidine 400.00 2,500.00L-Hydroxyproline 550.00L-Isoleucine 150.00 50.00L-Leucine 150.00 75.00L-Lysine•HCl 1,650.00 625.00L-Methionine 800.00 50.00L-Phenylalanine 150.00 150.00L-Proline 1,700.00 350.00L-Serine 250.00 550.00L-Threonine 350.00 175.00L-Tryptophan 100.00Insect MediumCat. No.ComponentSchneider’s04-351Liquid mg/lEurope onlyTC-100BE02-011Liquid mg/lL-Tyrosine 500.00L-Tyrosine•2Na•2H 2O 72.20L-Valine 300.00 100.00Vitaminsp-Aminobenzoic Acid 0.02d-Biotin 0.01D-Ca Pantothenate 0.02Choline Chloride 0.20Folic Acid 0.02i-Inositol 0.02Niacin 0.02Pyridoxine•HCl 0.02Riboflavin 0.02Thiamine•HCl 0.02Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com443
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and Sera9Iscove’s Modified Dulbecco’s (IMDM)Cat. No.Component12-722Liquidmg/l12-726Liquidmg/l12-915Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 165.00 165.00 165.00KCl 330.00 330.00 330.00KNO 30.076 0.076 0.076MgSO 4(anhyd.) 97.66 97.67 97.67NaCl 4,505.00 4,505.00 4,505.00NaHCO 33,024.00 3,024.00 3,024.00NaH 2PO 4•H 2O 125.00 125.00 125.00Na2SeO3•5H20 0.01 0.01 0.01Other ComponentsGlucose 4,500.00 4,500.00 4,500.00HEPES 5,958.00 5,958.00 5,958.00Phenol Red•Na 15.00 15.00 15.00Sodium Pyruvate 110.00 110.00 110.00Amino AcidsL-Alanine 25.00 25.00 25.00L-Arginine•HCl 84.00 84.00 84.00L-Asparagine•H 2O 28.40 28.40 28.40L-Aspartic Acid 30.00 30.00 30.00L-Cystine•2HCl 91.24 91.24 91.24L-Glutamic Acid 75.00 75.00 75.00L-Glutamine 584.00 584.00Glycine 30.00 30.00 30.00L-Histidine•HCl•H 2O 42.00 42.00 42.00L-Isoleucine 105.00 105.00 105.00L-Leucine 105.00 105.00 105.00L-Lysine•HCl 146.00 146.00 146.00L-Methionine 30.00 30.00 30.00L-Phenylalanine 66.00 66.00 66.00L-Proline 40.00 40.00 40.00L-Serine 42.00 42.00 42.00L-Threonine 95.00 95.00 95.00L-Tryptophan 16.00 16.00 16.00L-Tyrosine•2Na•2H 2O 104.20 104.20 104.20L-Valine 94.00 94.00 94.00Vitaminsd-Biotin 0.013 0.013 0.013D-Ca Pantothenate 4.00 4.00 4.00Choline Chloride 4.00 4.00 4.00Folic Acid 4.00 4.00 4.00i-Inositol 7.20 7.20 7.20Nicotinamide 4.00 4.00 4.00Pyridoxine•HCl 4.00 4.00 4.00Riboflavin 0.40 0.40 0.40Thiamine•HCl 4.00 4.00 4.00Vitamin B12 0.013 0.013 0.013Iscove, N.N. and F. Melchers (1978) J. of Exptl.Med.147: 923–933.444North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedL-15 (Leibovitz)McCoy’s 5AMcCoy’s 5ACat. No.Component12-6692X Liquidmg/l12-700Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 280.00 140.00KCl 800.00 400.00KH 2PO 4120.00 60.00MgCl 2anhyd.) 187.34 93.67MgSO 4(anhyd.) 195.46 97.70NaCl 16,000.00 8,000.00Na2HPO4 (anhyd.) 380.00 190.00Other ComponentsD( + ) Galactose 1,800.00 900.00Phenol Red•Na 10.00Sodium Pyruvate 1,100.00 550.00Amino AcidsDL-Alanine 900.00 450.00L-Arginine 1,000.00 500.00L-Asparagine 500.00 250.00L-Cysteine 240.00 120.00Glycine 400.00 200.00L-Histidine 500.00 250.00DL-Isoleucine 500.00 250.00Cat. No.Component12-168Liquidmg/l12-688Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 100.00 100.00KCl 400.00 400.00MgSO 4•7H 2O 200.00 200.00NaCl 5,460.00 6,460.00NaHCO 32,200.00 2,200.00NaH 2PO4 • H 2O 580.00 580.00Other ComponentsBactopeptone 600.00 600.00Glucose 3,000.00 3,000.00Glutathione (reduced) 0.50 0.50HEPES 5,957.40Phenol Red•Na 10.00 10.00Amino AcidsL-Alanine 13.36 13.36L-Arginine • HCl 42.14 42.14L-Asparagine • H 2O 45.03 45.03L-Aspartic Acid 19.97 19.97L-Cysteine • HCl • H 2O 35.12 35.12L-Glutamic Acid 22.07 22.07Cat. No.Component12-168Liquidmg/l12-688Liquidmg/lVitaminsp-Aminobenzoic Acid 1.00 1.00Ascorbic Acid 0.50 0.50d-Biotin 0.20 0.20D-Ca Pantothenate 0.20 0.20Choline Chloride 5.00 5.00Folic Acid 10.00 10.00i-Inositol 36.00 36.00Nicotinamide 0.50 0.50Nicotinic Acid 0.50 0.50Pyridoxal•HCl 0.50 0.50Pyridoxine•HCl 0.50 0.50Riboflavin 0.20 0.20Thiamine•HCl 0.20 0.20Vitamin B12 2.00 2.00McCoy, T.A., M. Maxwell, and P.F. Kruse, Jr.(1959) Proc. Soc. Exptl. Biol. Med. 100:115–118.Technical Information / Media, Reagents and SeraL-Leucine 250.00 125.00L-Glutamine 219.15 219.15L-Lysine 150.00 75.00Glycine 7.51 7.51DL-Methionine 300.00 150.00L-Histidine • HCl • H 2O 20.96 20.96DL-Phenylalanine 500.00 250.00Hydroxy L • Proline 19.67 19.67L-Serine 400.00 200.00L-Isoleucine 39.36 39.36DL-Threonine 1,200.00 600.00L-Leucine 39.36 39.36L-Tryptophan 40.00 20.00L-Lysine • HCl 36.54 36.54L-Tyrosine•2Na•2H 2O 370.00 370.00L-Methionine 14.92 14.92DL-Valine 400.00 200.00L-Phenylalanine 16.52 16.52VitaminsL-Proline 17.27 17.27DL-Ca Pantothenate 2.00 1.00L-Serine 26.28 26.28Choline Chloride 2.00 1.00L-Threonine 17.87 17.87Folic Acid 2.00 1.00L-Tryptophan 3.06 3.06i-Inositol 4.00 2.00L-Tyrosine 18.12 18.12Nicotinamide 2.00 1.00Pyridoxine•HCl 2.00 1.00L-Valine 17.57 17.579Riboflavin-5-PO 4•Na 0.20 0.10Thiamine•Monophosphate2.00 1.00Leibovitz:, A. (1963) Am. J. Hyg. 78:173–180.Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com445
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and Sera9Medium 199Cat. No.Component12-109Liquidmg/l12-117Liquidmg/l12-118Liquidmg/l12-119Liquidmg/lInorganic SaltsCaCl 2•2H 2O 186.00 265.00 186.00 265.00Fe(NO 3) 3•9H 2O 0.72 0.72 0.72 0.72KCl 400.00 400.00 400.00 400.00KH 2PO 460.00 60.00MgSO 4•7H 2O 200.00 200.00 200.00 200.00NaCl 8,000.00 5,800.00 7,000.00 6,800.00NaHCO 31,400.00 2,200.00 1,400.00 2,200.00Na 2HPO 4•7H 2O 90.00 90.00NaH2PO4•H2O 140.00 140.00Other ComponentsAdenine Sulfate 10.00 10.00 10.00 10.00Adenosine0.20 0.20 0.20 0.20MonophosphateAdenosine1.00 1.00 1.00 1.00Triphosphate•2 NaDeoxyribose 0.50 0.50 0.50 0.50Glucose 1,000.00 1,000.00 1,000.00 1,000.00Glutathione (reduced) 0.05 0.05 0.05 0.05Guanine • HCl 0.30 0.30 0.30 0.30HEPES 5,957.40 5,957.40Hypoxanthine 0.30 0.30 0.30 0.30Phenol Red • Na 15.00 15.00 15.00 15.00Ribose 0.50 0.50 0.50 0.50Sodium Acetate • 3H 2O 83.00 83.00 83.00 83.00Thymine 0.30 0.30 0.30 0.30Tween 80 4.90 4.90 4.90 4.90Uracil 0.30 0.30 0.30 0.30Xanthine 0.30 0.30 0.30 0.30Amino AcidsL-Alanine 25.00 25.00 25.00 25.00L-Arginine • HCl 70.00 70.00 70.00 70.00L-Aspartic Acid 30.00 30.00 30.00 30.00L-Cysteine • HCl • H 2O 0.10 0.10 0.10 0.10L-Cystine 20.00 20.00 20.00 20.00L-Glutamic Acid 67.00 67.00 67.00 67.00L-Glutamine 100.00 100.00 100.00 100.00Glycine 50.00 50.00 50.00 50.00L-Histidine • HCl • H 2O 22.00 22.00 22.00 22.00Hydroxy L • Proline 10.00 10.00 10.00 10.00L-Isoleucine 20.00 20.00 20.00 20.00L-Leucine 60.00 60.00 60.00 60.00L-Lysine • HCl 70.00 70.00 70.00 70.00L-Methionine 15.00 15.00 15.00 15.00L-Phenylalanine 25.00 25.00 25.00 25.00L-Proline 40.00 40.00 40.00 40.00L-Serine 25.00 25.00 25.00 25.00Medium 199Cat. No.Component12-109Liquidmg/l12-117Liquidmg/l12-118Liquidmg/l12-119Liquidmg/lL-Threonine 30.00 30.00 30.00 30.00L-Tryptophan 10.00 10.00 10.00 10.00L-Tyrosine 40.00 40.00 40.00 40.00L-Valine 25.00 25.00 25.00 25.00Vitaminsp-Aminobenzoic 0.05 0.05 0.05 0.05AcidAscorbic Acid 0.05 0.05 0.05 0.05d-Biotin 0.01 0.01 0.01 0.01Calciferol 0.10 0.10 0.10 0.10D-Ca Pantothenate 0.01 0.01 0.01 0.01Cholesterol 0.20 0.20 0.20 0.20Choline Chloride 0.50 0.50 0.50 0.50Folic Acid 0.01 0.01 0.01 0.01i-Inositol 0.05 0.05 0.05 0.05Menadione 0.01 0.01 0.01 0.01Nicotinamide 0.025 0.025 0.025 0.025Nicotinic Acid 0.025 0.025 0.025 0.025Pyridoxal • HCl 0.025 0.025 0.025 0.025Pyridoxine • HCl 0.025 0.025 0.025 0.025DL-Tocopheryl0.01 0.01 0.01 0.01Phosphate Na 2Riboflavin 0.01 0.01 0.01 0.01Thiamine • HCl 0.01 0.01 0.01 0.01Vitamin A Acetate 0.14 0.14 0.14 0.14Morgan, J.F., H.J. Morton, and R.C. Parker(1950) Proc. Soc. Exptl. Biol.Med. 73:1–8.446North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedMinimum Essential Medium (MEM)Cat. No.Component04-719Liquidmg/l06-174Liquidmg/l12-125Liquidmg/l12-127Liquidmg/l12-136Liquidmg/l12-137Liquidmg/l12-169Liquidmg/l12-611Liquidmg/l15-611Powdermg/l12-662Liquidmg/l12-668Liquid 2Xmg/lInorganic SaltsCaCl 2•2H 2O 265.00 186.00 265.00 186.00 265.00 265.00 264.87 265.00 530.00KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 800.00KH 2PO 460.00 60.00MgCl 2•6H 2O 200.00MgSO 4(anhyd.) 97.67MgSO 4•7H 2O 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 400.00NaCl 6500.00 6800.00 6800.00 8000.00 5800.00 7000.00 6800.00 6800.00 6800.00 6800.00 13600.00NaHCO 32000.00 2200.00 2200.00 350.00 2200.00 350.00 2200.00 2200.00 2200.00 4400.00NaH 2PO 4(anhyd.) 121.74NaH 2PO 4•H 2O 1327.00 140.00 140.00 140.00 140.00 140.00 140.00 280.00NaH2PO4•7H2O 90.00 90.00Other ComponentsGlucose 2000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 2000.00HEPES 5957.40 5957.50Lipoic Acid 0.20Phenol Red•Na 10.00 10.00 10.00 20.00 10.00 20.00 10.00 10.00 10.20 10.00Sodium Pyruvate 110.00 110.00Amino AcidsL-Alanine 8.90 25.00 8.90L-Arginine•HCl 126.00 126.40 126.40 126.40 126.40 126.40 126.40 126.40 126.98 126.40 252.80L-Asparagine 13.21L-Asparagine•H 2O 13.21 50.00L-Aspartic Acid 13.30 30.00 13.30Cysteine•HCl•H 2O 100.00L-Cystine•2HCl 32.40 31.29L-Cystine 24.00 24.00 24.00 24.00 24.00 24.00 24.00 24.00 48.00L-Glutamic Acid 14.70 75.00 14.70L-Glutamine 294.00 292.00 292.00 292.00Glycine 7.50 50.00 7.50L-Histidine, free base 31.00L-Histidine•HCl•H 2O 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 84.00L-Isoleucine 52.00 52.40 52.40 52.40 52.40 52.40 52.40 52.40 52.00 52.40 104.80L-Leucine 52.00 52.40 52.40 52.40 52.40 52.40 52.40 52.40 52.00 52.40 104.80L-Lysine, free base 58.00L-Lysine•HCl 73.00 73.00 73.00 73.00 73.00 73.00 73.00 72.46 73.00 146.00L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 30.00L-Phenylalanine 32.00 33.00 33.00 33.00 33.00 33.00 33.00 33.00 32.00 33.00 66.00L-Proline 11.50 40.00 11.50L-Serine 10.50 25.00 10.50L-Threonine 48.00 47.60 47.60 47.60 47.60 47.60 47.60 47.60 48.00 47.60 95.20L-Tryptophan 10.00 10.20 10.20 10.20 10.20 10.20 10.20 10.20 10.00 10.20 20.40L-Tyrosine 36.20 36.20 36.20 36.20 36.20 36.20 36.20 36.20 72.40L-Tyrosine•2Na 51.90L-Tyrosine•2Na•2H 2O 47.00L-Valine 46.00 46.80 46.80 46.80 46.80 46.80 46.80 46.80 46.00 46.80 93.60Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com447
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and SeraMinimum Essential Medium (MEM)Cat. No.Component04-719Liquidmg/l06-174Liquidmg/l12-125Liquidmg/l12-127Liquidmg/l12-136Liquidmg/l12-137Liquidmg/l12-169Liquidmg/l12-611Liquidmg/l15-611Powdermg/l12-662Liquidmg/lVitaminsAscorbic Acid 50.00d-Biotin 0.10D-Ca Pantothenate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00Choline Chloride 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00Folic Acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 4.00Nicotinamide 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00Pyridoxal•HCl 1.00 1.00 1.00 1.00 2.00Pyridoxine•HCl 1.00 1.00 1.00 1.00 1.00 1.00Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.20Thiamine•HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00Vitamin B12 1.3312-668Liquid 2Xmg/l9448North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedMinimum Essential Medium (MEM)Cat. No.Component12-684Liquid 10Xmg/l12-739Liquidmg/lBE02-002Liquidmg/lInorganic SaltsCaCl 2•2H 2O 2,650.00 264.92 265.00Fe(NO 3) 3•9H 2O 0.10KCl 4,000.00 400.00 400.00MgSO 4(anhyd.) 97.67MgSO 4•7H 2O 2000.00 200.00NaCl 68,000.00 6,400.00 6,800.00NaHCO 32,750.00 2,200.00NaH 2PO 4(anhyd.) 107.83NaH2PO4•H2O 1400.00 124.00 140.00Other ComponentsGlucose 10,000.00 4,500.00 1,000.00HEPESLipoic Acid 0.20Phenol Red • Na 100.00 15.30 10.00Sodium Pyruvate 110.00Amino AcidsL-Alanine 25.00L-Arginine • HCl 1,264.00 42.00 126.40L-Asparagine • H 2O 50.00L-Aspartic Acid 30.00Cysteine • HCl • H 2O 100.00L-Cystine • 2HCl 31.00L-Cystine 240.00 24.00L-Glutamic Acid 75.00L-Glutamine 292.00L-Alanyl-L-Glutamine434.00(UltraGlutamine 1)Glycine 50.00L-Histidine • HCl • H 2O 420.00 21.00 42.00L-Isoleucine 524.00 52.40 52.40L-Leucine 524.00 52.40 52.40L-Lysine • HCl 730.00 73.10 73.00L-Methionine 150.00 15.00 15.00L-Phenylalanine 330.00 33.00 32.00L-Proline 40.00L-Serine 25.00L-Threonine 476.00 47.60 47.60L-Tryptophan 102.00 8.00 10.20L-Tyrosine 362.00 36.20L-Tyrosine • 2Na • 2H 2O 52.90L-Valine 468.00 46.80 46.80Minimum Essential Medium (MEM)Cat. No.Component12-684Liquid 10Xmg/l12-739Liquidmg/lBE02-002Liquidmg/lVitaminsAscorbic Acid 50.00d-Biotin 0.10D-Ca Pantothenate 10.00 2.00 1.00Choline Chloride 10.00 2.00 1.00Folic Acid 10.00 2.00 1.00i-Inositol 20.00 3.60 2.00Nicotinamide 10.00 2.00 1.00Pyridoxine • HCl 10.00 2.00 1.00Riboflavin 1.00 0.20 0.10Thiamine • HCl 10.00 2.00 1.00Vitamin B12 1.33RibonucleosidesAdenosine 10.00Cytidine 10.00Guanosine 10.00Uridine 10.00Thymidine 10.00Deoxyribonucleosides2´ Deoxyadenosine 10.002´ Deoxycytidone HCl 11.002´ Deoxyguanosine 10.002´Deoxythymidine 10.00Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com449
Formulations for BioWhittaker Classical MediaContinuedNCTC 109 MediumNCTC 109 MediumRichter’s CM MediumTechnical Information / Media, Reagents and Sera9Cat. No.Component12-923Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 200.00KCl 400.00MgSO 4(anhyd.) 100.00NaCl 6,800.00NaHCO 32,200.00NaH 2PO 4•H 20 140.00Other ComponentsCocarboxylase (TPP) 1.00Coenzyme A (CoA) 2.50Deoxyadenosine 10.00Deoxycytidine • HCl 10.00Deoxyguanosine 10.00Diphosphopyridine7.00Nucleotide•4H 2OFlavin Adenine Dinucleotide • 2Na 1.00D-Glucosamine • HCl 3.85Glucose 1,000.00D-Glucuronolactone 1.80Glutathione (reduced) 10.005-Methylcytosine • HCl 0.16Phenol Red • Na 20.00Sodium Acetate • 3H 2O 50.00Sodium Glucuronate • H 2O 1.80Thymidine 10.00Triphosphopyridine Nucleotide • Na 1.00Tween 80 5.40Uridine 5-Triphosphate•3Na•2H2O 1.00Amino AcidsL-Alanine 31.48L-Amino Butyric Acid 5.51L-Arginine • HCl 31.16L-Asparagine • H 2O 9.19L-Aspartic Acid 9.91L-Cysteine • HCl • H 2O 288.57L-Cystine 10.49L-Glutamic Acid 8.26L-Glutamine 135.70Glycine 13.51L-Histidine • HCl • H 2O 26.65Hydroxy L • Proline 4.09L-Isoleucine 18.04L-Leucine 20.44L-Lysine • HCl 38.43L-Methionine 4.44Cat. No.Component12-923Liquidmg/lL-Proline 6.13L-Serine 10.75L-Taurine 4.18L-Threonine 18.93L-Tryptophan 17.50L-Tyrosine 16.40L-Valine 25.00Vitaminsp-Aminobenzoic Acid 0.125Ascorbic Acid 50.00d-Biotin 0.025Calciferol 0.250D-Ca Pantothenate 0.030Choline Chloride 1.250Folic Acid 0.025i-Inositol 0.125Menadione 0.025Niacinamide 0.063Niacin 0.063Pyridoxine • HCl 0.063Pyridoxal • HCI 0.063DL-Tocopherol phosphate • Na 20.025Riboflavin 0.025Thiamine • HCl 0.025Vitamin A Acetate 0.275Vitamin B12 10.00McQuilkin, W.T., V.J. Evans, and W.R. Earle(1957) J. Nat. Canc. Inst. 19:885–907.Cat. No.Component04-648Liquidmg/lInorganic SaltsCaCl 2•2H 20 264.90FeCl 3•6H 2O 0.54KCl 400.00MgCl 2•6H 2O 183.00MgSO 4•7H 2O 25.00NaCl 6,800.00NaHCO 32,200.00NaH 2PO 4•H 20 150.00ZnSO4•7H2O 0.14Other ComponentsGlucose 2,000.00Linoleic Acid 80.00Lipoic Acid 0.20Phenol Red • Na 10.00Putrescine • 2HCl 0.16Sodium Pyruvate 110.00Amino AcidsL-Arginine • HCl 127.00L-Asparagine • H 2O 60.00L-Cystine 24.00L-Glutamine 584.00L-Histidine • HCl • H 2O 42.00L-Isoleucine 52.00L-Leucine 131.20L-Lysine • HCl 72.60L-Methionine 15.00L-Phenylalanine 32.00L-Serine 42.00L-Threonine 48.00L-Tryptophan 10.00L-Tyrosine 36.00L-Valine 46.00Vitaminsd-Biotin 0.10Choline Chloride 56.00D-Ca Pantothenate 1.00Folic Acid 2.20i-Inositol 36.00Nicotinamide 1.00Pyridoxine • HCl 1.00Riboflavin 0.10Thiamine • HCl 1.00Vitamin B12 1.36L-Ornithine • HCl 9.41L-Phenylalanine 16.53450North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedRPMI MediumCat. No.Component09-774Liquid mg/l12-115Liquid mg/l12-167Liquid mg/l12-918Liquid mg/l12-702Liquid mg/l04-525Liquid mg/l15-702Powder mg/lBE12-752Liquid mg/lBE12-11 5 /U1Liquid mg/lBE12-70 2 /U1Liquid mg/lInorganic SaltsCa(NO 3) 2•4H 2O 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00MgSO 4(anhyd.) 48.83MgSO 4•7H 2O 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00NaCl 5,000.00 5,000.00 6,000.00 6,000.00 6,000.00 6,000.00 6,000.00 6,000.00 5,000.00 6,000.00NaHCO 32,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00Na 2HPO 4800.49Na2HPO4•7H2O 1,512.00 1,512.00 1,512.00 1,512.00 1,512.00 1,512.00 1,512.00 1,512.00 1,512.00Other ComponentsGlucose 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00 2,000.00Glutathione (reduced) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00HEPES 5,957.50 5,957.40 5,957.50MOPS 34,535.00Penicillin G Potassium 100(units/ml)Phenol Red • Na 5.00 5.00 5.00 5.00 5.00 5.10 5.00 5.00 5.00Streptomycin50.00(mcg/ml)Amino AcidsL-Arginine 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00 200.00L-Arginine • HCl 241.86L-Asparagine • H 2O 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00L-Aspartic Acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Cystine • 2HCl 65.19L-Cystine 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00L-Glutamic Acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Glutamine 300.00 300.00 300.00 300.00 300.00 300.00L-Alanyl-L-Glutamine446.00 446.00(UltraGlutamine 1)Glycine 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00L-Histidine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00L-Histidine • HCl • H 2O 20.27Hydroxy L • Proline 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Isoleucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00L-Leucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00 50.00L-Lysine • HCl 40.00 40.00 40.00 40.00 40.00 40.00 40.00 40.00 40.00 40.00L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00L-Phenylalanine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00L-Proline 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Serine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00L-Threonine 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Tryptophan 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00L-Tyrosine 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00L-Tyrosine • 2Na 28.83L-Valine 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com451
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and Sera9RPMI MediumCat. No.Component09-774Liquid mg/l12-115Liquid mg/l12-167Liquid mg/l12-918Liquid mg/l12-702Liquid mg/lMoore, G.E., R.E. Gerner, and H.A. Franklin (1967) J. Am. Med. Assoc. 199:87 – 92.04-525Liquid mg/l15-702Powder mg/lBE12-752Liquid mg/lBE12-11 5 /U1Liquid mg/lVitaminsp-Aminobenzoic Acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00d-Biotin 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20D-Ca Pantothenate 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25Choline Chloride 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00Folic Acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00i-Inositol 35.00 35.00 35.00 35.00 35.00 35.00 35.00 35.00 35.00 35.00Nicotinamide 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Pyridoxine • HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Riboflavin 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20Thiamine • HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Vitamin B12 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005Williams’ Medium ECat. No.ComponentEurope OnlyBE12-761Liquid mg/lBE02-019Liquid mg/lInorganic SaltsCaCl 2(anhyd.) 200.00 200.00CuSO 4•5H 2O 0.0001 0.0001Fe(NO 3) 3•9H 2O 0.0001 0.0001KCl 400.00 400.00MgSO 4(anhyd.) 400.00 400.00MgSO • 47H 2O 200.00 200.00MnCl 2•4H 2O 0.0001 0.0001NaCl 6,800.00 6,800.00NaHCO 32,200.00 2,200.00NaH 2PO 4•H 2O 140.00 140.00ZnSO4•7H2O 0.0002 0.0002Other ComponentsGlucose 2,000.00 2,000.00Glutathione (reduced) 0.05 0.05Linoeic Acid Methyl Ester 0.03 0.03Phenol Red Na 10.00Sodium Pyruvate 25.00 25.00Tween 80 1.836 1.836Amino AcidsL-Alanine 90.00 90.00L-Arginine 50.00 50.00L-Asparagine H 2O 20.00 20.00L-Aspartic Acid 30.00 30.00L-Cysteine 40.00 40.00L-Cystine 20.00 20.00L-Glutamic Acid 50.00 50.00Glycine 50.00 50.00Williams’ Medium ECat. No.ComponentEurope OnlyBE12-761Liquid mg/lBE12-70 2 /U1Liquid mg/lBE02-019Liquid mg/lL-Histidine 15.00 15.00L-Isoleucine 50.00 50.00L-Leucine 75.00 75.00L-Lysine HCl 87.50 87.50L-Methionine 15.00 15.00L-Phenylalanine 25.00 25.00L-Proline 30.00 30.00L-Serine 10.00 10.00L-Threonine 40.00 40.00L-Thryptophan 10.00 10.00L-Tyrosine 35.00 35.00L-Valine 50.00 50.00VitaminsAscorbic Acid 2.00 2.00d-Biotin 0.50 0.50D-Ca Pantothenate 1.00 1.00Choline Chloride 1.50 1.50Ergocalciferol 0.10 0.10Folic Acid 1.00 1.00i-Inositol 2.00 2.00Menadione Na bisulfite 0.01 0.01Nicotinamide 1.00 1.00Pyridoxal HCl 1.00 1.00DL-Tocopherol phosphate Na 20.01 0.01Riboflavin 0.10 0.10Thiamine HCl 1.00 1.00Vitamin A Acetate 0.10 0.10Vitamin B12 0.20 0.20452North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Formulations for BioWhittaker Classical MediaContinuedWaymouth’s MB 752/1 MediumCat. No.ComponentBE12-738Liquid mg/lInorganic SaltsCaCl 2(anhyd.) 90.61KCl 150.00KH 2PO 480.00MgCl 2(anhyd.) 112.56MgSO 4(anhyd.) 97.67NaCl 6,000.00NaHCO 32,240.00Na2HPO4 (anhyd.) 300.00Other ComponentsGlucose 5,000.00Glutathione (reduced) 15.00Hypoxanthine Na 29.00Phenol Red Na 10.20Amino AcidsL-Arginine HCl 75.00L-Aspartic Acid 60.00Cysteine HCl H 2O 100.29L-Cystine 2 HCl 20.00L-Glutamic Acid 150.00L-Glutamine 350.00Glycine 50.00L-Histidine HCl H 2O 164.10L-Isoleucine 25.00L-Leucine 50.00L-Lysine HCl 240.00L-Methionine 50.00L-Phenylalanine 50.00L-Proline 50.00L-Threonine 75.00L-Thryptophan 40.00L-Tyrosine 2Na 2H 2O 57.66L-Valine 65.00VitaminsAscorbic Acid 17.50d-Biotin 0.02D-Ca Pantothenate 1.00Choline Chloride 250.00Folic Acid 0.40i-Inositol 1.00Nicotinamide 1.00Pyridoxine HCl 1.00Riboflavin 1.00Thiamine HCl 10.00Vitamin B12 0.20Lymphocyte SeparationMediumCat. No.ComponentMEM Vitamin SolutionCat. No.Component17-829Liquid mg/lFicoll 400 62,000.00*Sodium Diatrizoate 104,000.00Specific Gravity1.077 g/ml*Slight adjustments may be required toobtain the specified specific gravityMEM Non-essentialAmino AcidsCat. No.Component13-114Liquid mg/lL-Alanine 890.00L-Asparagine 1,321.00L-Aspartic Acid 1,330.00L-Glutamic Acid 1,470.00Glycine 750.00L-Proline 1,150.00L-Serine 1,050.0013-607Liquid mg/lD-Ca Pantothenate 100.00Choline Chloride 100.00Folic Acid 100.00i-Inositol 200.00Nicotinamide 100.00Pyridoxine • HCl 100.00Riboflavin 10.00Thiamine•HCl 100.00Technical Information / Media, Reagents and Sera9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com453
Formulations for BioWhittaker Classical MediaContinuedTechnical Information / Media, Reagents and SeraAntibiotic SolutionsCat. No.Component/units09-757Liquid17-51817-528Liquid17-519Liquid17-602LiquidAmphotericin B (mg/l) 25.00 250.00Gentamicin sulfate (mg/l) 50,000.00 10,000.00 10,000.00L-Glutamine (mg/l) 29,200.00NaCl (mg/l) 8,500.00 8,500.00 8,500.00 8,500.00Potassium Penicillin (units/ml) 20,000.00 10,000.00 5,000.00 25,000.00 25,000.00 10,000.00Streptomycin sulfate (mcg/ml) 20,000.00 10,000.00 5,000.00 25,000.00 25,000.00 10,000.00Balanced Salt Solutions (EBSS and HBSS)Cat. No.Component– EBSS Europe only – – HBSS –10-502Liquid mg/l02-027Liquid mg/l04-315Liquid mg/l10-508Liquid mg/l10-527Liquid mg/l17-603Liquid17-718Liquid10-543Liquid mg/l17-719Liquid10-547Liquid mg/lInorganic SaltsCaCl 2•2H 2O 265.00 265.00 186.0 186.0KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00KH 2PO 460.00 60.00 60.00 60.00 60.00MgSO 4•7H 2O 200.00 200.00 200.00 200.00NaCl 6,800.00 6,800.00 8,000.00 8,000.00 8,000.00 8,000.00 8,000.00NaHCO 32,200.00 1,800.00 350.00 350.00 350.00 350.00 350.00Na 2HPO 4•2H 2O 158.00 158.00Na2HPO4•7H2O 90.00 90.00 90.00 90.00 90.00Other ComponentsGlucose (anhyd.) 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00Glucose • H 2O 1,100.00 1,100.00HEPES 4,766.00Phenol Red•Na 10.60 10.60 20.00 20.0017-745Liquid17-836Liquid02-012LiquidDulbecco’s Phosphate Buffered Saline (DPBS)Cat. No.Component04-479Liquid mg/l17-512Liquid mg/l17-513Liquid mg/l17-515Liquid 10X mg/l9Inorganic SaltsCaCl 2•2H 2O 130.00 130.00KCl 200.00 200.00 200.00 2,000.00KH 2PO 4200.00 200.00 200.00 2,000.00MgCl 2•6H 2O 100.00 100.00MgCl 2(anhyd.)NaCl 8,000.00 8,000.00 8,000.00 80,000.00Na 2HPO 4Na2HPO4•7H2O 2,160.00 2,160.00 2,160.00 21,600.00Other ComponentsGlucose 1,000.00Sodium Pyruvate 36.00454North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Cell Culture Reagent FormulationsMiscellaneous Buffer SolutionsCat. No.ComponentDGV Buffer10-539Liquidmg/lACKLysingBuffer10-548Liquidmg/lHEPESBuffer17-737Liquidmg/lTL HEPESSolution04-616Liquidmg/lUltraSalineA12-747Liquidmg/lInorganic SaltsCaCl 2(anhyd.) 20.00CaCl 2•2H 2O 300.00EDTA, Na 23.72KCl 240.00 223.68KHCO 31001.00MgCl 2•6H 2O 100.00MgSO 4•7H 2O 120.00NaCl 8,500.00 8,500.00 6,660.00 7,130.00NaHCO 3168.00NaH 2PO 4•H 2O 47.60Na 2HPO 4•7H 2O 268.07NH4Cl 8,023.50Other ComponentsBSA Fraction V 3,000.00DL-Lactic Acid (Na1.86 mlSalt) (60% syrup)Gelatin 600.00Glucose 10,000.00 720.64HEPES 238,300.00 2,400.00 7,150.00Phenol Red (0.5%1.0 mlin DPBS)Sodium Veronal 380.00Veronal 580.00Veronal Buffer (5X)Cat. No. 12-624ComponentLiquid mg/lCaCl 2•2H 20 185.00MgCl 2•6H 20 840.00NaCl 42,500.00Sodium Barbital 1,870.00Barbital 2,870.00Trypsin and Versene® SolutionsCat. No.Component17-160Liquidmg/l17-161Liquidmg/l17-711Liquidmg/lEuropeonlyBE02-007ELiquidmg/lGlucose 1,000.00 1,000.00KCl 400.00 400.00 200.00KH 2PO 460.00 200.00NaCl 8,000.00 8,000.00 8,000.00 9,000.00NaHCO 3580.00Na 2HPO 4•7H 2O 90.00 2,170.00Phenol Red • Na 2.00Trypsin 25,000.00 500.00 5,000.00Versene®(EDTA)•2Na•2H 2O200.00 200.00 2,000.00Technical Information / Media, Reagents and SeraReagents –––– Europe Only – –––Cat. No.CWomponentProHTBE17-855Liquidmg/lUltra Glutamine IBE17-60 5 /U1Liquidmg/lUltra Glutamine IIBE04-684Liquidmg/lHATBE02-024Liquidmg/lInorganic SaltsNaCl 8,500.00 8,500.00Other ComponentsAminopterin 8.80Hypoxanthine 1,927.00 680.00Thymidine 100.00 194.00Amino AcidsL-Arginine HClL-CystineL-Alanyl-L-Glutamine43,444.00(UltraGlutamine I)L-Glycyl-L-Glutamine44,240.00H 2O (UltraGlutamine II)Phosphate Buffered SalineCat. No.Component04-409Liquidmg/l17-516Liquidmg/l17-517Liquid 10xmg/lBE02-017Liquidmg/lInorganic SaltsKCl 280.00KH 2PO 4144.00 144.00 1,440.00 260.00NaCl 9,000.00 9,000.00 90,000.00 8,120.00Na 2HPO 4795.00 795.00 7950.00Na 2HPO 4•2H 2O 1,170.00Titriplex III (EDTA Na 2) 460.00LiCl 426.709Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com455
Electrophoresis and AnalysisFrequently Asked Questions – Nucleic AcidTechnical Information / Electrophoresis and Analysis9Electrophoresis and AgaroseQ. What buffer conditions give me best resolution foragarose electrophoresis?A. For small DNA fragments (15,000 bp), 1X TAE Bufferenhances separation of large DNA. Since TAE has alower buffering capacity, it may be necessary either torecirculate the buffer, or periodically mix the bufferbetween the anodal and cathodal chambers whenelectrophoresing for an extended period of time. Thetime to buffer depletion can vary <strong>with</strong> the volts/hourand the size of chamber used.Buffer depth over the gel should be 3 to 5 mm deep.Less buffer, and you risk the chance of the gel dryingout. Excessive buffer will decrease the resistance of thecircuit between the anode and cathode, which results ina decreased voltage gradient through the gel. Thiscauses inefficient DNA mobility, excessive heating, andband distortion.Q. How should I cast my gels to get the best resolution?A. We usually cast gels 3 mm to 4 mm thick. The gelvolume needed can easily be estimated by measuringthe surface area of the casting chamber, thenmultiplying by the gel thickness. Thinner gels can becast on GelBond® Support Film, and/or cast in a verticalapparatus.The thickness of the comb in the direction of theelectrical field can also profoundly affect the resolution.A thin comb (1 mm) will result in sharper DNA bands.With too thick a comb, the separated DNA bands will bequite broad.Q. My DNA bands are sometimes wavy, but usually only inone or two lanes. What causes this?A. Dried agarose on the comb teeth is a frequent cause ofthis problem. Prior to casting your gel, check the combteeth for residual dried agarose. If not removed, this willattach to the newly cast agarose and fracture the wellupon comb removal. This is usually not observable untilthe gel is on the transilluminator. Additionally, caremust be taken during comb removal, particularly <strong>with</strong>low melting temperature agaroses. Well integrity maybe maintained in these agaroses by pre-chilling the gelto 4°C for 30 minutes and/or by flooding the gel <strong>with</strong>cold buffer prior to removing the comb.Q. How much DNA should I load per well?A. The amount to load per well is variable. What is mostimportant is how much DNA there is in the bands youwish to resolve. The least amount of DNA that can beconsistently detected <strong>with</strong> ethidium bromide is about10 ng. The most DNA you can have in a band and still geta sharp, clean band on an ethidium bromide stained gelis about 100 ng. These amounts will be less on gelsstained <strong>with</strong> more sensitive stains such as GelStar®Stain. On a GelStar® Stained Gel it is possible to detectas little as 20 pg dsDNA.The optimal amount of DNA to load in the well iscalculated by the fraction of the total DNA which is in theband of interest. If you are unsure of how much DNA ispresent, load varying amounts in several lanes ifpossible.The optimal amount of DNA to load in the well may becalculated by the fraction of the total DNA which is in theband of interest, represented by the following:Fragment of interest (kbp)X 100 = % DNA in band of interestSize of DNA sample (kbp)NOTE: The most DNA compatible <strong>with</strong> a clean sharp band is approximately100 ng.For example:The size of your DNA sample is 48.5 kbp and when runon the gel 8 fragments are separated. Your fragment ofinterest is 2.3 kbp.Calculation:2.3 kbp48.5 kbpX 100 =4.7% DNA in fragment of interestIf you load 1 µg of DNA, then 4.7% of the 1 µg of loadedsample will appear in your fragment of interest (47 ng).To further increase the sharpness of the bands, use aFicoll® Based Loading Buffer, such as Lonza DNALoading Buffer (Cat. No. 50655) instead of sucrosebasedor glycerol-based loading buffers. The use oflower molecular weight glycerol will allow DNA to streamup the sides of the well before electrophoresis whichresults in U-shaped bands.Loading buffer that is too high in ionic strength cancause the bands to be fuzzy. In the ideal situation, theDNA sample should be suspended in the same solutionas the running buffer. If this is not possible, use asample buffer <strong>with</strong> a lower ionic strength than therunning buffer.456North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Frequently Asked QuestionsContinuedQ. At what voltage should I run an agarose gel?A. We recommend running agarose gels at 4–10 volts/cm(cm is determined by measuring the interelectrodedistance, not the gel length) under normal horizontalelectro phoretic conditions. If the voltage is too high,band streaking, especially for DNA >15 kb, may result.When the voltage is too low, the mobility of small(15 kb)DNA fragments using conventional horizontal electrophoresis.The best separations in this instance areobtained at a voltage gradient of
Frequently Asked Questions – Protein AnalysisTechnical Information / Electrophoresis and AnalysisPAGEr Precast GelsQ. Which PAGEr Precast Gels will fit my gel chamber?A. PAGEr Precast Gels are available in 9 cm × 10 cm and10 cm × 10 cm sizes and fit most standard mini-verticalsystems. Some chambers may require modificationsfor optimal fit <strong>with</strong> PAGEr Precast Gels.Standard Vertical SystemsPAGEr Minigel ChamberBio-Rad® Mini-PROTEAN® II, Mini-PROTEAN® 3 ,Mini-PROTEAN® Tetra, Mini-PROTEAN® Dodeca andReady Gel® Cell Systems.Reverse the inner core gasket so the flat side faces outward.Novex® XCell SureLock® Mini-Cell or XCell IIRequest the spacer for the XCell SureLock® Mini-Cell Chamberfrom Scientific Support, (Cat. No. 59900).FisherBiotech® Vertical Minigel FBVE121,Owl Separations Systems Wolverine P82Chamber comes <strong>with</strong> 2 sets of wedges. Use the thinnerwedges for the PAGEr Gold Gels.FisherBiotech® Vertical Minigel FB-VE101,Owl Separations Systems Penguin Model P8DSRequest adaptor for these chambers from Scientific Support,(Cat. No. 59902).Hoefer® Mighty Small (SE250, SE260)If using SE250 replace the buffer chamber <strong>with</strong> a ’Deep lowerbuffer chamber for the SE260’, order number 80-6148-78,from GE Healthcare.Daiichi 2, ISS chambersHoefer® Mighty Small (SE260)EC 120 Mini Vertical Gel SystemBiometra® Mini V ChamberCBS Scientific MGV System,(10 cm × 8 cm units)Sigma-Aldrich Mini Techware(11.3 cm × 10 cm units)Zaxis System 2000Hoefer® Mini VEPAGEr Gels9 cm × 10 cm or10 cm × 10 cm gels*9 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels*10 cm × 10 cm gels10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels*10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels9 cm × 10 cm or10 cm × 10 cm gels9 cm × 10 cm gels9 cm × 10 cm gels10 cm × 10 cm gels10 cm × 10 cm gels10 cm × 10 cm gels*Recommended for best fitstacking/resolving gel boundary. This stacking effectresults in superior resolution.Q. I would like to run a native or nondenaturing gel. Whatcan I use?A. PAGEr Precast Gels do not contain SDS or any otherdenaturing agents (e.g., DTT and b-ME). Additionally,you would use a Tris-Glycine Running Buffer that doesnot contain SDS.9Q: Do Lonza PAGEr Precast Gels contain a stacking gel?What is the purpose of the stacking gel?A. PAGEr Gold Precast Gels contain a 4% stacking gel,pH 8.6. The purpose of this stacking gel is to allow theproteins to accumulate and condense (i.e., stack) at the458North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Frequently Asked QuestionsContinuedProtein ElectrophoresisQ. How do I make the transfer, running, and samplebuffers?A. Tris-Glycine Gels (Tris-HCl Buffer System)Towbintransfer buffer (1X)Running buffer (1X)Sample buffer (1X)0.025 M Tris base 25 mM Tris Base 62.5 mM Tris-HCl, pH 6.80.192 M Glycine 192 mM Glycine 2% SDS*0.05–0.1% SDS* 0.1% SDS* 10% Glycerol20% Methanol 0.01% Bromophenol Blue2.5% bME(2-mercaptoethanol)**Omit for native proteins.For best results use Lonza AccuGENE Electrophoresis Buffers or useProSieve EX Running Buffer for fast denaturing protein separation.ProSieve EX Running Buffer is required <strong>with</strong> PAGEr EX Gels.Q. What is the difference between gradient vs. homogeneous(single concentration) gels? Which oneshould I use?A. Gradient gels are suitable for a wide range of sizeresolutions. A homogeneous or single concen tration gelis appropriate where the proteins of interest are knownto be <strong>with</strong>in a narrow size range.Q. How much protein should I load on the gel?A. Protein load levels will vary depending upon samplepurity and staining method used. For highly purifiedproteins, 0.5 μg to 5 μg protein per lane on a minigel isgenerally sufficient. Complete mixtures such as celllysates may require as much as 50 μg protein per lane.Protein Stain Detection LimitsProtein stainLower detection limit(protein/band)Coomassie® Blue Stain30 ngSilver Stain2 ngSYPRO® Red Protein Gel Stain4 ng–8 ngSYPRO® Ruby Protein Gel Stain2 ng–8 ngSYPRO® Tangerine Protein Gel Stain 4 ng–8 ngProSieve EX Safe Stain8 ng–15 ngNOTE: Limits are based on optimal detection methods for each stain.Q. What is the best membrane to use for Western blotting?A. Use this table to find a suitable membrane.Nitrocellulose PVDF NylonHydrophobic binding Hydrophobic binding Hydrophobic andelectrostatic bindingGeneral purpose membrane SDS tolerant Stable if bakedLow background High background High backgroundLow strength High strength High strengthBecomes brittle if baked Suitable for proteinsequencingLeast suitable forWestern transferQ. What are the benefits of using agarose for protein gelelectrophoresis?A. Protein electrophoresis in agarose gels is an alternativeapproach to using polyacrylamide gels and providesseveral benefits:––Separate high molecular weight proteins (>600 kDa)––Easy to prepare and handle––Efficient recovery of proteins––Excised proteins can be used to immunie animalsdirectly for antibody production––Non-toxic––Run gels using either a vertical or horizontal apparatusTechnical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com459
Agarose TypesTechnical Information / Electrophoresis and AnalysisThe appropriate choice of agarose depends on the size ofthe DNA to be analyzed and any subsequent manipulationsrequired. Gelling/melting temperatures, electroendoosmosisand gel strength are all important factors in choosing theright agarose for your application. Refer to page 462 foranalytical specifications of Lonza Agarose.Genetic Technology Grade (GTG) AgarosesOur Genetic Technology Grade (GTG) Agarose Productsare specially prepared and certified for demandingmolecular biology applications for nucleic acids, includingPCR amplified products. Our GTG Agarose quality tests gobeyond standard assays, such as DNase and RNase testing,to include enzymatic performance measurements. Ouradditional testing provides a more realistic index of overallproduct quality and reliability. You no longer need to screenagarose lots to find those that yield biologically active DNA.■■The following agaroses are GTG Certified:––SeaKem® GTG Agarose––SeaPlaque GTG Agarose (low melting temperatureagarose)––NuSieve GTG Agarose (low melting temperatureagarose)––SeaKem® Gold AgaroseMolecular Biology Grade AgarosesMolecular biology grade agaroses are suitable for generalanalytical separation of DNA.■■The following agaroses are considered molecularbiology grade agaroses:––MetaPhor Agarose––SeaKem® LE Agarose––NuSieve 3:1 Agarose––SeaPlaque Agarose (low melting temperature agarose)■■We screen our molecular biology grade agaroses for thefollowing parameters:––DNA binding––DNase and RNase activity––Gel background stainingFDA ListingOur agarose types are listed as Class 1 Medical Devicesunder registration number 1219614.■■We perform the following tests on GTG CertifiedAgaroses:––DNA binding––DNase and RNase activity––DNA resolution––Gel background-gel exhibits low backgroundfluorescence after ethidium bromide staining––In-gel cloning (low melt agarose)––In-gel restriction digestion (low melt agarose)––Restriction-ligation assay (SeaKem® GTG)9460North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Preparation of Agarose GelsSuggested Agarose Concentrations and Dye Migration InformationTable 1: Suggested Agaroses for Particular Applications Table 2: Properties of TAE and TBE Buffer SystemsSize Range(base pairs)Agarose Type20–800 MetaPhorAgarose50–1,000 NuSieve 3:1AgaroseNuSieve GTGAgarose1,000–10,000 SeaKem® GTGAgaroseSeaPlaqueGTG Agarose10,000 SeaKem® GoldAgaroseApplicationHigh resolution analysis;2% size differencesAnalysis and blotting;4%–6% size differences resolvedAnalysis and blotting; In-gel;6% size differences resolvedAnalysis and blotting; recoveryrequiredIn-gelAnalysisAgarose Analytical SpecificationsAgaroseMeltingTemperatureBufferTAE bufferTBE bufferSuggested Uses and CommentsUse when DNA is to be recoveredUse for electrophoresis of large (>12 kb) DNALow ionic strengthLow buffering capacity – recirculation may be necessaryfor extended electrophoretic timesUse for electrophoresis of small (
Technical Information / Electrophoresis and Analysis9Preparation of Agarose GelsContinuedTable 3: Suggested Agarose Concentrations for DNASizesSize range (base pairs) Final Agarose Concentration % (w/v)1X TAE buffer 1X TBE bufferSeaKem® LE and SeaKem® GTG Agarose1,000–23,000 0.60 0.50800–10,000 0.80 0.70400–8,000 1.00 0.85300–7,000 1.20 1.00200–4,000 1.50 1.25100–3,000 2.00 1.75NuSieve 3:1 Agarose500–1,000 3.0 2.0100–500 4.0 3.010–100 6.0 5.0MetaPhor Agarose150–800 2.0 1.8100–600 3.0 2.050–250 4.0 3.020–130 5.0 4.012,000 bp.Table 4: Migration of Double-stranded DNA in Relationto Bromophenol Blue (BPB) and Xylene Cyanol (XC) inAgarose Gels1X TAE Buffer % Agarose 1X TBE BufferXC BPB XC BPBSeaKem® LE and SeaKem® GTG Agarose24,800 2,900 0.30 19,400 2,85016,000 1,650 0.50 12,000 1,35010,200 1,000 0.75 9,200 7206,100 500 1.00 4,100 4003,560 370 1.25 2,500 2602,800 300 1.50 1,800 2001,800 200 1.75 1,100 1101,300 150 2.00 850 70NuSieve 3:1 Agarose950 130 2.50 700 70650 80 3.00 500 40350 40 4.00 250 20200 30 5.00 140 8120 20 6.00 90 4MetaPhor Agarose480 70 2.00 310 40200 40 3.00 140 35120 35 4.00 85 3085 30 5.00 60 15SeaPlaque and SeaPlaque GTG Agarose11,700 1,020 0.50 6,100 4004,000 500 0.75 2,850 2802,300 350 1.00 1,700 1801,500 200 1.25 1,000 1001,000 150 1.50 700 70700 100 1.75 500 50550 60 2.00 400 30320 30 2.50 250 10NuSieve GTG Agarose750 175 2.50 460 75400 120 3.00 210 35115
Preparation of Agarose GelsDissolving AgaroseAgarose undergoes a series of steps when it is dissolved:dispersion, hydration and melting/dissolution.Microwave Instructions for Gel Concentrations
Preparation of Agarose GelsContinuedTechnical Information / Electrophoresis and AnalysisMicrowave Instructions for Gel Concentrations ≥2% w/v1. Choose a beaker that is 2–4 times the volume of thesolution.2. Add room temperature or chilled buffer (for MetaPhorand NuSieve GTG Agarose) and a stir bar to the beaker.3. Sprinkle in the premeasured agarose powder while thesolution is rapidly stirred to prevent the formation ofclumps.4. Remove the stir bar if not Teflon®-coated.5. Soak the agarose in the buffer for 15 minutes beforeheating. This reduces the tendency of the agarosesolution to foam during heating.6. Weigh the beaker and solution before heating.7. Cover the beaker <strong>with</strong> plastic wrap.8. Pierce a small hole in the plastic wrap for ventilation. Foragarose concentrations >4%, the following additionalsteps will further help prevent the agarose solutionfrom foaming during melting/dissolution:8.1 Heat the beaker in the microwave oven on mediumpower for 1 minute.8.2 Remove the solution from the mircrowave.8.3 Allow the solution to sit on the bench for 15minutes.9. Heat the beaker in the microwave oven on mediumpower for 2 minutes.CAUTION: Any microwaved solution may becomesuperheated and foam over when agitated.Hot Plate Instructions for Preparing Agarose1. Choose a beaker that is 2–4 times the volume of thesolution.2. Add room temperature or chilled buffer (for MetaPhoror NuSieve GTG Agarose) and a stir bar to the beaker.3. Sprinkle in the premeasured agarose powder while thesolution is rapidly stirred to prevent the formation ofclumps.4. Weigh the beaker and solution before heating.5. Cover the beaker <strong>with</strong> plastic wrap.6. Pierce a small hole in the plastic wrap for ventilation.7. Bring the solution to a boil while stirring.8. Maintain gentle boiling until the agarose is dissolved(approximately 5–10 minutes).9. Add sufficient hot distilled water to obtain the initialweight.10. Mix thoroughly.11. Cool the solution to 60°C prior to casting.CAUTION: Always wear eye protection, and guardyourself and others against scalding solutions.910. Remove the beaker from the microwave oven.CAUTION: Use oven mitts when removing beaker frommicrowave, as container will be hot and may causeburns.11. Gently swirl to resuspend any settled powder and gelpieces.12. Reheat the beaker on high power for 1–2 minutes oruntil the solution comes to a boil.13. Hold at the boiling point for 1 minute or until all of theparticles are dissolved.14. Remove the beaker from the microwave oven.15. Gently swirl to mix the agarose solution thoroughly.16. After dissolution, add sufficient hot distilled watertoobtain the initial weight.17. Mix thoroughly.18. Cool the solution to 60°C prior to gel casting.464North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Preparation of Agarose GelsContinuedHorizontal Gel Casting Instructions1. Allow the agarose solution to cool to 60°C.2. While the agarose solution is cooling:2.1 Assemble the gel casting tray.2.2 Level the casting tray prior to pouring the agarosesolution.2.3 Check the comb(s)* teeth for residual driedagarose. Dried agarose can be removed byscrubbing the comb teeth <strong>with</strong> a lint-free tissuesoaked in hot distilled water.2.4 Allow a small space (approximately 0.5 mm–1 mm) between the bottom of the comb teeth andthe casting tray.3. Pour the agarose solution into the gel tray.4. Replace the comb(s).5. Allow the agarose to gel at room temperature for30 minutes.6. Low melting temperature agaroses and MetaPhorAgarose require an additional 30 minutes of gelling at4°C to obtain the best gel handling. The additionalcooling step is essential for obtaining fine resolution inMetaPhor Agarose.7. Once the gel is set, flood <strong>with</strong> running buffer.8. Slowly remove the comb.9. Place the gel casting tray into the electrophoresischamber.10. Fill the chamber <strong>with</strong> running buffer until the bufferreaches 3 mm–5 mm over the surface of the gel.11. Gently flush the wells out <strong>with</strong> electrophoresis bufferusing a Pasteur pipette to remove loose gel fragmentsprior to loading the samples.12. Load DNA and electrophorese.Voltage TableThe table below provides a quick reference for optimalvoltage for DNA electrophoresis.Recommended Voltages and Buffers Related to DNASize and ApplicationSize Voltage – – Buffer– –Recovery Analytical≤1 kb 5 V/cm TAE TBE1 kb to 12 kb 4–10 V/cm TAE TAE/TBE>12 kb 1–2 V/cm TAE TAEOptimal Electrophoretic TimeThe gel should be run until the band of interest has migrated40%–60% down the length of the gel (see the Dye MobilityTable). Band broadening resulting from dispersion anddiffusion results in a decrease in resolution in the lowerthird of the gel. Resolution may also be decreased in smallergels, since longer electrophoretic runs result in greaterseparation between two fragments.Technical Information / Electrophoresis and AnalysisThe thickness of the comb in the direction of the electricfield can affect the resolution. A thin comb (1 mm) willresult in sharper DNA bands. With a thicker comb, morevolume can be added to the well but the separated DNAbands may be broader.■■Materials––Horizontal electrophoresis apparatus––Combs––Pasteur pipette■■Reagents––Agarose solution––Electrophoresis buffer9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com465
Loading BuffersTechnical Information / Electrophoresis and AnalysisGel loading buffers serve three purposes in DNAelectrophoresis:––Increase the density of the sample: This ensures thatthe DNA will drop evenly into the well––Add color to the sample: Simplifies loading––Add mobility dyes: The dyes migrate in an electric fieldtowards the anode at predictable rates. This enablesone to monitor the electrophoretic processTypes of Loading BuffersAt least five loading buffers are commonly used for agarosegel electro phoresis. They are prepared as six-foldconcentrated solutions. If needed, 10X solutions of eachbuffer can also be prepared. Alkaline loading buffer is usedwhen performing alkaline gel electrophoresis.LoadingBuffer6X recipeSucrose-based40% (w/v) Sucrose0.25% Bromophenol Blue0.25% Xylene cyanol FFGlycerol-based30% Glycerol in distilled water0.25% Bromophenol Blue0.25% Xylene cyanol FFFicoll®-based 15% Ficoll® (Type 400)Polymer in distilled water0.25% Bromophenol Blue0.25% Xylene cyanol FFAlkaline300 mN NaOH6 mM EDTA18% Ficoll® (Type 400)Polymer in distilled water0.15% Bromocresol Green0.25% Xylene cyanol FFStorageTemperature4°C4°Croomtemperature4°CFicoll®-Based Loading BuffersTo increase the sharpness of DNA bands, use Ficoll®(Type 400) Polymer as a sinking agent instead of glycerol.The use of the lower molecular weight glycerol in the loadingbuffer allows DNA to stream up the sides of the well beforeelectrophoresis has begun and can result in a U-shapedband. In TBE gels, glycerol also interacts <strong>with</strong> borate, whichcan alter the local pH.Sample PreparationLoading buffer that is too high in ionic strength causesbands to be fuzzy and migrate through the gel atunpredictable rates. Ideally, the DNA sample should beresuspended in the same solution as the running buffer. Ifthis is not possible, use a sample buffer <strong>with</strong> a lower ionicstrength than the running buffer.9466North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Detection and Sizing of DNA in Agarose GelsFlashGel DNA and RNA MarkersSize Range 0.5 Kb – 9.0 kb 0.5 Kb – 9.0 kb 100 bp–3 kb 100 bp–4 kb 100 bp–1.5 kb 50 bp–1.5 kbLadder/marker RNA marker RNA marker DNA marker DNA marker Quant ladder DNA markerCat. No. 50575 50577 57034 50473 50475 57033Number of bands 10 6 6 8 5 8<strong>Catalog</strong> No.50473FlashGel DNAMarker100 bp–4 kb1.2%, 12 + 1(Standard)9.0 kb 9.0 kb 3,000 bp 4,000 bp 1,500 bp 1,500 bp6.0 kb 5.0 kb 1,500 bp 2,000 bp 800 bp 800 bp5.0 kb 3.0 kb 800 bp 1,250 bp 400 bp 500 bp4.0 kb 1.5 kb 500 bp 800 bp 250 bp 300 bp300 bp3.0 kb 1.0 kb 100 bp 500 bp 100 bp 200 bp2.5 kb 0.5 kb 300 bp 150 bp2.0 kb 200 bp 100 bp1.5 kb 100 bp 50 bp1.0 kb0.5 kb– 4,000 bp– 2,000 bp– 1,250 bp– 800 bp– 500 bp– 300 bp– 200 bp– 100 bp50475FlashGel DNAMarker100 bp–1.5 kb1.2%, 12 + 1(Quantitation)– 1,500 bp (30ng)– 800 bp (21ng)– 400 bp (15 ng)– 250 bp (7.5 ng)– 100 bp (3 ng)57033– 1,500 bp– 800 bp– 500 bp– 300 bp– 200 bp– 150 bp– 100 bp– 50 bpFlashGel DNAMarker50 bp–1.5 kb2.2%, 12 + 1(HighConcentration)57034– 3,000 bp– 1,500 bp– 800 bp– 500 bp– 300 bp– 100 bp– 3,000 bp– 1,500 bp– 800 bp– 500 bp– 300 bp– 100 bpFlashGel DNAMarker100 bp–3 kb1.2%, 16 + 1(Double-tier)50577– 9.0 Kb– 5.0 Kb– 3.0 Kb– 1.5 Kb– 1.0 Kb– 0.5 KbFlashGel RNAMarker0.5 kb–9 kb1.2%, 12 + 1 & 16+1(RNA)50575– 9 kb– 6 kb– 5 kb– 4 kb– 3 kb– 2.5 kb– 2 kb– 1.5 kb– 1 kb– 0.5 kbLonza RNA Marker0.5 bp–9 kb1.2%, 12 + 1(RNA)Technical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com467
Detection and Sizing of DNA in Agarose GelsContinuedTechnical Information / Electrophoresis and Analysis9Guide to Lonza Ladders and Markers – Size Range (Bold numbers indicate brighter bands)20 bp 20 bp Ext 100 bp 100 bp ext Tandem 500 bp QuantLadderRev QuantLadder50 bp –1000 bp50 bp –2500 bpStandard laddersCat. No. 50330 50320 50321 50322 NA 50323 50334 50335 50461 50631 50471SimplyLoad laddersCat. No. 50331 50326 50327 50328 50333 50329 50336 50337 NA NA NANumber of 25 50 100 30 21 16 5 5 9 13 9fragmentsSize range 20 bp–500 bp20 bp–1,000 bp100 bp–1,000 bp10 bp–3,000 bp100 bp–12 kb500 bp–8 kb100 bp–1,000 bp100 bp–1,000 bp50 bp–1,000 bp50 bp–2,500 bp1 kb –10 kb500 bp 1,000 bp 1,000 bp 3,000 bp 12 kb 8,000 bp 1,000 bp 1,000 bp 1,000 bp 2.5 kb 10 kb480 bp 980 bp 900 bp 2,900 bp 11 kb 7,500 bp 700 bp 700 bp 700 bp 2 kb 7 kb460 bp 960 bp 800 bp 2,800 bp 10 kb 7,000 bp 500 bp 500 bp 525 bp 1.5 kb 5 kb440 bp 940 bp 700 bp 2,700 bp 9 kb 6,500 bp 200 bp 200 bp 500 bp 1250 bp 4 kb420 bp 920 bp 600 bp 2,600 bp 8 kb 6,000 bp 100 bp 100 bp 400 bp 1 kb 3 kb400 bp 900 bp 500 bp 2,500 bp 7 kb 5,500 bp 300 bp 700 bp 2.5 kb380 bp 880 bp 400 bp 2,400 bp 6 kb 5,000 bp 200 bp 525 bp 2 kb360 bp 860 bp 300 bp 2,300 bp 5 kb 4,500 bp 100 bp 500 bp 1.5 kb340 bp 840 bp 200 bp 2,200 bp 4 kb 4,000 bp 50 bp 400 bp 1 kb320 bp 820 bp 100 bp 2,100 bp 3 kb 3,500 bp 300 bp300 bp 800 bp 2,000 bp 2 kb 3,000 bp 200 bp280 bp 780 bp 1,900 bp 1 kb 2,500 bp 100 bp260 bp 760 bp 1,800 bp 900 bp 2,000 bp 50 bp240 bp 740 bp 1,700 bp 800 bp 1,500 bp220 bp 720 bp 1,600 bp 700 bp 1,000 bp200 bp 700 bp 1,500 bp 600 bp 500 bp180 bp 680 bp 1,400 bp 500 bp160 bp 660 bp 1,300 bp 400 bp140 bp 640 bp 1,200 bp 300 bp120 bp 620 bp 1,100 bp 200 bp100 bp 600 bp 1,000 bp 100 bp80 bp 580 bp 900 bp60 bp 560 bp 800 bp40 bp 540 bp 700 bp20 bp 520 bp 600 bp500 bp 500 bp480 bp 400 bp460 bp 300 bp440 bp 200 bp420 bp 100 bp400 bp380 bp360 bp340 bp320 bp300 bp280 bp260 bp240 bp1 kb –10 kb468North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Detection and Sizing of DNA in Agarose GelsContinued20 bp 20 bp Ext 100 bp 100 bp ext Tandem 500 bp QuantLadder220 bp200 bp180 bp160 bp140 bp120 bp100 bp80 bp60 bp40 bp20 bpRev QuantLadder50 bp –1000 bp50 bp –2500 bp1 kb –10 kbTechnical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com469
Detecting DNA <strong>with</strong> GelStar®, SYBR® Green I or II Nucleic Acid Gel StainsTechnical Information / Electrophoresis and AnalysisFollow the Steps Below to Stain DNA afterElectrophoresis1. Remove the concentrated stock solution of GelStar® orSYBR® Green Stain from the freezer and allow thesolution to thaw at room temperature.2. Spin the solution in a microcentrifuge to collect the dyeat the bottom of the tube.3. Dilute the 10,000X concentrate to a 1X working solution(1 µl per 10 ml), in a pH 7.5–8.5 buffer, in a clear plasticpolypropylene container. Prepare enough stainingsolution to just cover the top of the gel.4. Remove the gel from the electrophoresis chamber.5. Place the gel in staining solution.6. Gently agitate the gel at room temperature.7. Stain the gel for 15–30 minutes. The optimal stainingtime depends on the thickness of the gel, concentrationof the agarose, and the fragment size to be detected.Longer staining times are required as gel thickness andagarose concentration increase.8. Remove the gel from the staining solution and view<strong>with</strong> a 300 nm UV transilluminator, CCD camera or DarkReader® Transilluminator (Clare Chemical <strong>Research</strong>,Inc.). GelStar® and SYBR® Green Stained Gels do notrequire destaining. The dyes fluorescence yield ismuch greater when bound to DNA than when in solution.Follow this Procedure When Including GelStar® Stain inthe Agarose Gel1. Remove the concentrated stock solution of GelStar®Stain from the freezer and allow the solution to thaw.2. Spin the solution in a microcentrifuge tube.3. Prepare the agarose solution (see pages 444–445).4. Once the agarose solution has cooled to 70°C, add thestain by diluting the stock 1:10,000 into the gel solutionprior to pouring the gel (1 µl per 10 ml).5. Slowly swirl the solution.6. Pour the gel into the casting tray (see page 446).7. Load your DNA onto the gel.8. Run the gel.9. Remove the gel from the electrophoresis chamber.10. View <strong>with</strong> a 300 nm UV transilluminator, CCD camera orDark Reader® Transilluminator (Clare Chemical <strong>Research</strong>,Inc.). GelStar® Stained gels do not require destaining.The dye’s fluorescence yield is much greater whenbound to DNA than when in solution.Staining Vertical Gels <strong>with</strong> GelStar® andSYBR® Green StainsIncorporating GelStar® and SYBR® Green Stains into the gelor prestaining the DNA for use in a vertical format is notrecommended. The dye binds to glass or plastic plates andDNA may show little to no signal. Gels should be poststainedas described in the previous section.Follow this procedure when staining vertical gels <strong>with</strong>GelStar® or SYBR® Green Stain:9Follow steps 1–4, above.11. Open the cassette and leave the gel in place on oneplate.12. Place the plate, gel side up in a staining container.13. Gently pour the stain over the surface of the gel.14. Stain the gel for 5–15 minutes.15. View <strong>with</strong> a 300 nm UV transilluminator, CCD camera orDark Reader® Transilluminator (Clare Chemical<strong>Research</strong>, Inc.) GelStar® or SYBR® Green Stained gelsdo not require destaining. The dye’s fluorescence yieldis much greater when bound to DNA than when insolution.470North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Detecting DNA <strong>with</strong> GelStar®, SYBR® Green I or II Nucleic Acid Gel StainsContinuedVisualization by PhotographyGels stained <strong>with</strong> GelStar® and SYBR® Green Stains exhibitnegligible background fluorescence, allowing long filmexposures when detecting small amounts of DNA. Use theappropriate photographic filter for the stain.Suggested film types and photographic conditions:Poloaroid® Film f-stop Exposure TimeType 57 or 667 4.5 0.5–2 secondsType 55 4.5 15–45 secondsVisualization by Image Capture SystemFor the best results and optimal sensitivity, visualizeGelStar® Stained Gels on The Dark Reader® Transilluminator(Clare Chemical <strong>Research</strong>, Inc.) GelStar® and SYBR® GreenStains are compatible <strong>with</strong> most CCD and video imagingsystems. Due to variations in the filters for these systems,you may need to purchase a new filter. Contact yoursystems manufacturer.Stain Emission (nm) Excitation (nm)GelStar® Stain 527 493SYBR® Green I Stain 521 494SYBR® Green II Stain 513 497Application Notes––The fluorescent characteristics of GelStar® and SYBR®Green Stains make them compatible <strong>with</strong> argon ionlasers.––These stains are removed from double-stranded DNA bystandard procedures for ethanol precipitation of nucleicacids.––Gels previously stained <strong>with</strong> ethidium bromide cansubsequently be stained <strong>with</strong> GelStar® or SYBR® GreenStain following the standard protocol for post-staining.––The inclusion of GelStar® and SYBR® Green Stains incesium chloride density gradient plasmid preparationsis not recommended. The effect of the dye on thebuoyant density of DNA is unknown.––These stains do not appear to interfere <strong>with</strong> enzymaticreactions.––We recommend the addition of 0.1% to 0.3% SDS in theprehybridization and hybridization solutions whenperforming Southern blots on gels stained <strong>with</strong> thesedyes.––Double-stranded DNA-bound GelStar® or SYBR® GreenStain fluoresces green under UV transillumination. Gelsthat contain DNA <strong>with</strong> single-stranded regions mayfluoresce orange rather than green.DecontaminationStaining solutions should be disposed of by passing throughactivated charcoal followed by incineration of the charcoal.For absorption on activated charcoal, consult Sambrook, etal., pp. 6.16–6.19, (1989). Follow state and local guidelinesfor decontamination and disposal of nucleic acid stainingsolutions.Technical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com471
Detecting DNA <strong>with</strong> Ethidium BromideTechnical Information / Electrophoresis and AnalysisEthidium bromide is a fluorescent dye which detects bothsingle- and double-stranded DNA. However, the affinity forsingle-stranded DNA is relatively low compared to doublestrandedDNA. Ethidium bromide-stained DNA is detectedby ultraviolet radiation. At 254 nm, UV light is absorbed bythe DNA and transmitted to the dye; at 302 nm, and366 nm, UV light is absorbed by the bound dye itself. In bothcases, the energy is re-emitted at 590 nm in the red-orangeregion of the visible spectrum.ProcedureFor optimal resolution, sharpest bands and lowestbackground, stain the gel <strong>with</strong> ethidium bromide followingelectrophoresis.Ethidium bromide can also be included in the gel andelectrophoresis buffer (0.5 µg/ml) <strong>with</strong> only a minor loss ofresolution. The electrophoretic mobility of DNA will bereduced by approximately 15%.Follow the Steps below to Stain DNA AfterElectrophoresis1. Prepare enough working solution of ethidium bromide.(0.5–1 µg/ml of ethidium bromide in distilled water orgel buffer) to cover the surface of the agarose gel.2. Remove the gel from the electrophoresis chamber.3. Submerge the gel for 20 minutes in the ethidium bromidesolution.4. Remove the gel from the solution.5. Submerge the gel for 20 minutes in a new containerfilled <strong>with</strong> distilled water.6. Repeat in fresh distilled water.7. Gels can be viewed <strong>with</strong> a hand-held or tabletop UVlight. For gel concentrations of 4% or greater, thesetimes may need to be doubled. If after destaining thebackground is still too high, continue to destain.■■Reagents––Ethidium bromide stock solution (10 mg/ml)––Electrophoresis buffer or distilled waterCaution: Materials and methods shown here presenthazards to the user and the environment. Refer to thesafety information on page 459 before beginningthese procedures.Follow the Steps Below When Including EthidiumBromide in the Agarose Gel8. Prepare agarose solution (see pages 444-445).9. While the agarose solution is cooling, add ethidiumbromide to a final concentration of 0.1 to 0.5 µg/ml tothe solution.10. Gently swirl the solution.11. Pour the gel into the casting tray.12. Add ethidium bromide to the running buffer to a finalconcentration of 0.5 µg/ml.13. Load and run the gel.14. Destain the gel by submerging the gel in distilled waterfor 20 minutes.15. Repeat in fresh distilled water.16. Gels can be viewed <strong>with</strong> a hand-held or tabletop UV lightduring or after electrophoresis.Decontamination of Ethidium Bromide SolutionsDecontamination of ethidium bromide solutions isdescribed in Sambrook, et al., pp. 6.16–6.17 (1989).Follow local guidelines and regulations for ethidium bromidedecontamination and disposal.9■■Materials––Staining vessel larger than gel––UV transilluminator––Magnetic stir plate––Magnetic stir bar472North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Recovery of DNA from Agarose GelsTips for Increasing DNA Recovery Efficiency fromAgarose GelsThis section discusses various tips which will increase theefficiency of recovery of DNA from agarose gels in allrecovery techniques.Choosing the Appropriate Agarose for RecoveryWhen recovering DNA, the choice of agarose is one of themost important factors. To avoid recovery altogether, onecan choose to perform in-gel reactions.We offer Genetic Technology Grade (GTG) Products thatare specially prepared for demanding molecular biologyapplications. See pages 257–259. (See table below for ouragaroses and compatible recovery techniques.)Buffer TypesWhen recovering DNA from agarose gels, 1X Tris-acetate(TAE) Buffer is recommended for electrophoresis.Casting and DNA Loading Tips––Prepare the gel in 1X TAE Buffer––Do not cast the gel <strong>with</strong> ethidium bromide––Cast a gel 3–4 mm thick––Use a comb ≤1 mm thick––Load no more than 100 ng of DNA per bandStaining and Recovery TipsWhen recovering DNA from agarose gels, we recommendthe following:––Stain the gel for 15–20 minutes––Destain the gel in distilled water for two, 20-minutewashes––Do not expose the DNA to UV light for any longer than1 minute; long exposure of DNA to UV light can nickthe DNA––The addition of 1 mM guanosine or cytidine to the geland electrophoresis buffer is effective in protecting DNAagainst UV-induced damage––Cut the smallest gel slice possibleIt is possible to avoid staining samples which will be usedfor recovery by running an additional lane containing asmall amount of your sample immediately next to themolecular weight marker. However, DNA is damaged by UVlight in the absence of ethidium bromide so keep exposureto UV light as brief as possible. Cut the lanes containing themarker and the small amount of the sample from the rest ofthe gel and stain. To recover the preparative loading, line upthe stained portion of the gel <strong>with</strong> the unstained portion.Check by placing on UV transilluminator and cut out thearea that lines up <strong>with</strong> your sample on the unstained portionof the gel.The FlashGel System for Recovery, (page 270) offers anon-UV alternative for DNA recovery.Technical Information / Electrophoresis and AnalysisReferenceGrundemann, D. and Schomig, E., BioTechniques 21(5): 898–903,1996.Lonza Agaroses and Compatible Recovery TechniquesIn-Gel β-Agarase Phenol/Chloroform Recovery Columns Electroelution Freeze/SqueezeSeaKem® GTG Agarose ■ ■ ■ ■SeaPlaque GTG Agarose ■ ■ ■ ■ ■ ■NuSieve GTG Agarose ■ ■ ■ ■ ■ ■MetaPhor Agarose ■ ■ ■ ■SeaPlaque Agarose ■ ■ ■ ■ ■9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com473
Recovery of DNA from Agarose GelsTechnical Information / Electrophoresis and AnalysisPhenol/Chloroform Extraction of DNA from Agarose GelsCompatible Agaroses––SeaPlaque GTG Agarose (certified and tested for therecovery of DNA)––NuSieve GTG Agarose (certified and tested for therecovery of DNA)––SeaPlaque AgaroseTipsRecovery failures when extracting DNA from agaroseusing phenol/chloroform most often result from eitherextracting too large a piece of agarose, or precipitatingagarose along <strong>with</strong> the DNA at the ethanol precipitationstep. To address these difficulties, we recommend thefollowing:––No more than 200 mg (200 µl) of agarose should beextracted in a single tube; if your gel slice containingthe DNA is larger than this, separate it into smallerpieces, then combine the extracted solutions prior toethanol precipitation––Ethanol precipitation of agarose can be avoided bychilling the extracted solution on ice for 15 minutes, thencentrifuging the sample(s) in a cold room for 15 minutesat maximum speed in a microcentrifuge prior to addingsalts and ethanol. The supernatant is then carefullydecanted, and the DNA in the supernatant is precipitatedfollowing standard protocols––Not useful for large DNA (>10kb). Vortexing will shearthe DNAEthanol Precipitation of DNA Recovered fromAgarose GelsTips––Prior to adding salts and ethanol, precipitation ofagarose can be avoided by chilling the supernatant onice for 15 minutes, then centrifuging the sample(s) in acold room for 15 minutes at maximum speed in amicrocentrifuge. The supernatant is then carefullydecanted, and the DNA in the supernatant is ethanolprecipitated following standard protocols––Ethanol precipitations should be incubated at roomtemperature <strong>with</strong> ammonium acetate rather thansodium acetate in order to decrease the likelihood ofcoprecipitation of agarose-oligosaccharides <strong>with</strong> theDNA or RNA9474North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Protein Separation in Polyacrylamide GelsBuffers for Protein ElectrophoresisThe Laemmli Buffer System (Tris-Glycine) is a discontinuousbuffer system, widely used for fine resolution of abroad molecular weight range of proteins. In this system,the gel is prepared <strong>with</strong> Tris-HCl Buffer and the Tris-glycineis used as the running buffer.In the Tris-Tricine Buffer System, tricine replaces glycine inthe running buffer. The result is more efficient stacking anddestacking, and higher resolution of proteins and peptides<strong>with</strong> lower molecular weights (under 10 kDa – 15 kDa). Forfast reliable separation in Laemmli gels such as PAGEr Goldprecast gels or when using PAGEr EX precast gels usProSieve EX Running Buffer. See pageBuffer PreparationTris-Glycine SDS Buffer, pH 8.310x Stock solutiong/l for 10X Stock solution0.25 M Tris base 30.3 g Tris Base1.92 M Glycine 144.0 g Glycine1.0% SDS* Adjust volume to 1 liter <strong>with</strong>distilled water(1X = 25 mM Tris base, 192 mM Glycine, 0.1% SDS*)*Omit SDS if running native proteins.Tris-Tricine SDS Buffer, pH 8.310x Stock solutiong/l for 10X Stock solution1 M Tris base 121.1 g Tris base1 M Tricine 179.0 g Tricine1.0% SDS* Adjust volume to 1 liter <strong>with</strong>distilled water(1X = 100 mM Tris base, 100 mM Tricine, 0.1% SDS*)*Omit SDS if running native proteins.2X Tris-Glycine SDS Sample Buffer2X concentrateamount to add for 2X concentrate126 mM Tris-HCl, pH 6.8 2.5 ml of 0.5 M Tris-HCl, pH 6.820% Glycerol 2 ml Glycerol4% SDS 4 ml of 10% SDS0.005% Bromophenol blue 0.5 ml of 0.1% Bromophenol blueAdjust volume to 10 ml <strong>with</strong> distilled water(1X = 63 mM Tris-HCl, 10% glycerol, 2% SDS, 0.0025% Bromophenol blue,2.5% βME)Technical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com475
Protein Separation in Polyacrylamide GelsContinuedTechnical Information / Electrophoresis and AnalysisLoading and Running Proteins on Polyacrylamide GelsProtein load levels will vary depending upon sample purityand staining method used. For highly purified proteins, 0.5µg to 5 µg protein per lane on a minigel is generallysufficient. Complete mixtures such as cell lysates mayrequire as much as 50 µg protein per lane. The table belowprovides lower detection limits for protein detection.Protein Stain Detection LimitsProtein stainLower Detection Limit(protein/band)Coomassie® Blue Stain100 ngSilver Stain1 ngSYPRO® Orange Protein Gel Stain 1 ng–2 ngSYPRO® Red Protein Gel Stain1 ng–2 ngSYPRO® Tangerine Protein Gel Stain 4 ng–8 ngProSieve Safe Stain8 ng–15 ngNOTE: Limits are based on optimal detection methods for each stain.Optimal Voltage and Power SettingsTris-glycine polyacrylamide minigels are typically run atconstant voltage between 125–200 volts. Duringelectrophoresis, the current drops and heat decreases.Voltage set too high, or not limited causes excessiveheating, resulting in band distortion and potential damageto the gel and apparatus. Constant voltage allows the samevoltage to be used <strong>with</strong> multiple gels in an apparatus. Gelthickness is not a factor when using constant voltage. Forlarge format gels, a constant current setting <strong>with</strong> a voltagelimit set slightly higher (5 volts) than the expected voltagefor the run may also be used to maintain sample velocity.By substituting 1X ProSieve EX Running Buffer forTris‐glycine SDS one can run polyacrylamide mini-gels at ahigher voltage of 200–250V for much faster runs whilemaintaining resolution of bands. See pageUsing PAGEr EX Gels and 1X ProSieve EX Running Buffermaximizes speed and resolution. See page?Optimal Electrophoretic TimeThe gel should be run until the bromophenol blue dye hasmigrated to the bottom of the gel. Gel running times aredependent upon the buffer system used, the length of thegel and the polyacrylamide concentration. Typicallyminigels will take approximately 30–90 minutes to run,whereas large format gels may take as long as five hours torun.By substituting 1X ProSieve EX Running Buffer for Tris-Glycine SDS and running at a higher voltage of 200–250Vthe time can be cut to as little as 15 minutes whilemaintaining resolution of bands. See pageUsing PAGEr EX Gels and 1X ProSieve EX Running Buffermaximizes speed and resolution. See page9476North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Blotting Proteins from Polyacrylamide GelsIntroductionProtein transfer efficiency in blotting applications isdependent upon multiple factors, including gel percentage,gel thickness, protein size, transfer conditions (e.g., bufferand voltage), and type and quality of membrane. To achieveoptimal transfer efficiency, transfer conditions must beadjusted to address these varying factors.Choosing the Appropriate MembraneNitrocellulose PVDF NylonHydrophobic binding Hydrophobic binding Hydrophobic and electrostatic bindingGeneral purpose membrane SDS tolerant Stable if bakedLow background High background High backgroundLow strength High strength High strengthBecomes brittle if baked Suitable for protein sequencing Least suitable for Western transferTransfer SolutionsFormula for Towbin Transfer Solution1X Working SolutionAmount for 1X Working Solution25 mM Tris base 30.3 g Tris base192 mM Glycine 144.1 g Glycine0.1% SDS 10.0 gAdjust volume to 8 liters <strong>with</strong> distilled water.Measure, but do not adjust pH; it should be approximately 8.2 to 8.420% Methanol 2 l MethanolAdjust volume to 10 liters <strong>with</strong> distilled waterTechnical Information / Electrophoresis and AnalysisIt may be necessary to lower the concentrations ofmethanol, SDS or both to obtain the optimal balance oftransfer and binding efficiency. The table below outlines theeffects that SDS and methanol have on protein transfer.SDSImproves transfer of proteins >60 kDaDecreases binding efficiencyNot compatible <strong>with</strong> nylon membranesInclude 0.1%–0.2% in transfer bufferMethanolImproves binding efficiencyDecreases transfer efficiencyDo not soak gel in transfer buffer prior to blottingInclude 20% in transfer buffer9For rapid efficient transfer of proteins from PAGEr Gold andPAGEr EX Gels or other Laemmli precast mini gels use 1XProSieve EX Western Transfer Buffer. It adds speed, is easyto use and does not contain methanol. See pageEurope – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com477
Electrophoretic TheoryElectrophoretic ParametersTechnical Information / Electrophoresis and Analysis9During electrophoresis, one of the parameters is heldconstant and the other two are allowed to vary as theresistance of the electrophoretic system changes. Invertical systems, the resistance of the gel increases ashighly conductive ions like Cl are electrophoresed out of thegel. As these ions are removed from the gel, the current iscarried by less conductive ions like glycine, borate, acetate,etc. Under normal conditions in horizontal systems, there islittle change in resistance. However, <strong>with</strong> high voltage orextended runs in horizontal systems, resistance candecrease.IntroductionThere are advantages and disadvantages for setting each ofthe critical parameters as the limiting factor inelectrophoresis. Sequencing gels are usually run atconstant wattage to maintain a uniform temperature.Agarose and acrylamide gels for protein and DNA separationare run at constant voltage or constant current.Constant WattageIn a vertical system when wattage is held constant, thevelocity of the samples will decrease because the current,which is in part carried by the DNA, decreases to compensatefor the increase in voltage. The generation of heat willremain uniform.If the current should decrease disproportionately (from abuffer problem, a buffer leak or a hardware problem), thepower supply will increase the voltage to compensate.Since voltage and current vary over time at a constantwattage, it is not possible to predict mobility of samplesfrom the calculation of watt-hours.Volts & watts20015010025201510mAConstant CurrentWhen the current is held constant, the samples will migrateat a constant rate. Voltage and wattage will increase as theresistance increases, resulting in an increase in heatgeneration during the run.If a break occurs in the system, such as a damaged lead orelectrode or a buffer leak, the resistance of the gel will begreatly increased. This will cause a large increase in wattageand voltage resulting in the generation of excessive heat. Itis Constant even Curren possible for the system to get hot enough to boil, orstart the apparatus to scorch or burn.Volts & watts20015010050000 1 2 3 4 5 6 7Time (hours)mA Volts WattsConstant VoltageWhen voltage is set constant, current and wattage willdecrease as the resistance increases, resulting in adecrease of heat and DNA migration.Since the heat generated will decrease, the margin of safetywill increase over the length of the run. If a problem developsand the resistance increases dramatically, the current andwattage will fall since the voltage cannot increase. Even ifthe apparatus fails, the worst that is likely to happen is thatthe resistance will increase so much that the power supplywill not be able to compensate, and it will shut off.Volts & watts200150252015105706050mAmA50000 1 2 3 4 5 6 7Time (hours)mA Volts Watts51005040302010ReferenceRickwood, D. and Hames, B.D., Gel Electrophoresis of Nucleic Acids: APractical Approach, IRL Press Limited, 1982.000 1 2 3 4 5 6 7Time (hours)mA Volts Watts478North America – Customer Service: 800 638 8174 (toll free); order.us@lonza.com; Scientific Support: 800 521 0390 (toll free) scientific.support@lonza.com
Safety and Environmental PrecautionsIn general, working <strong>with</strong> nucleic acids and proteins does notpresent significant hazards to humans so long asprecautions are taken to protect against certain harmfulmaterials.Throughout the Technical Information section are referencesto materials and methods which are hazardous to humansand the environment. Specific hazards and protection stepsare summarized here, and it is recommended that trainedSpecific Chemical HazardsEthidium BromideEthidium bromide (EtBr) is a known mutagen and asuspected carcinogen. Care should be taken to preventexposure. Recommended personal protective equipmentincludes: nitrile gloves, lab coats, and safety glasses. Useethidium bromide solutions in a well ventilated area, andprevent inhalation of vapors. Electrophoresis tanks shouldbe kept covered during electrophoresis of gels containingethidium bromide. Ethidium bromide powder should behandled in a fume hood.Decontamination and disposal of ethidium bromide shouldbe done according to local government and institutionalregulations.GelStar® and SYBR® Green Nucleic Acid Gel StainsGelStar® and SYBR® Green Stains contain a componentwhich can penetrate cells (including skin), and is a potentialmutagen. Therefore, care should be taken to preventexposure. Recommended personal protective equipmentincludes: (non nitrile) gloves, lab coats, and safety glasses.Decontamination and disposal of these stains should bedone according to local government and institutionalregulations.users follow these precautions when performing theoperations outlined in this manual.These precautions are not a substitute for proper health andsafety training, nor are they a substitute for the userinstitution’s standards and procedures, or localgovernmental requirements. In each case, the user shouldbe aware of and follow local guidelines for handling anddisposal of these materials.Formaldehyde and FormamideFormaldehyde and formamide are known carcinogens, andexposure should be limited to as low as feasible. Gloves,safety glasses (or goggles when pouring liquid), and labcoats should be worn. Operations using formaldehyde orformamide should be conducted <strong>with</strong> the use of respiratoryprotection, or in a fume hood or other well ventilated area toprevent inhalation.Disposal of these materials should be done according tolocal government and institutional regulations.DMSODimethyl Sulfoxide (DMSO) can penetrate cells and DNA,and is a carrier of other substances in solutions into thosecells. Care should be taken to prevent exposure, includingthe use of non nitrile gloves (natural rubber arerecommended), safety glasses and lab coat.Disposal of these materials should be done according tolocal government and institutional regulations.PhenolPhenol is toxic by inhalation, ingestion, and contact. It willburn eyes. Appropriate gloves, safety glasses (or goggles),and lab coats are required. Proper ventilation shouldbe utilized.Disposal of these materials should be done according tolocal government and institutional regulations.Technical Information / Electrophoresis and Analysis9Europe – Customer Service: +32 87 321 611; order.europe@lonza.com; Scientific Support: +32 87 321 611; scientific.support.eu@lonza.comInternational – Customer Service: +1 301 898 7025; Scientific Support: scientific.support@lonza.com479
www.lonza.comContact InformationNorth AmericaCustomer Service: 800 638 8174 (toll free)order.us@lonza.comScientific Support: 800 521 0390 (toll free)scientific.support@lonza.comEuropeCustomer Service: +32 87 321 611order.europe@lonza.comScientific Support: +32 87 321 611scientific.support.eu@lonza.comInternationalContact your local Lonza distributorCustomer Service: +1 301 898 7025Fax: +1 301 845 8291scientific.support@lonza.comInternational OfficesAustralia + 61 3 9550 0883Austria0800 201 538 (toll free)Belgium + 32 87 321 611Brazil + 55 11 2069 8800Denmark808 83 159 (toll free)France0800 91 19 81 (toll free)Germany0800 182 52 87 (toll free)India +91 40 4123 4000Ireland1 800 654 253 (toll free)Italy800 789 888 (toll free)Japan + 81 3 6264 0660Luxemburg +32 87 321 611Norway800 16 557 (toll free)Poland + 48 781 120 300Singapore + 65 6521 4379Spain900 963 298 (toll free)Sweden020 790 220 (toll free)Switzerland 0800 83 86 20 (toll free)The Netherlands 0800 022 4525 (toll free)United Kingdom 0808 234 97 88 (toll free)Lonza Walkersville, Inc. – Walkersville, MD 21793Unless otherwise noted, all trademarks herein are marks of theLonza Group Ltd or its affiliates. The information contained hereinis believed to be correct and corresponds to the latest stateof scientific and technical knowledge. However, no warranty ismade, either expressed or implied, regarding its accuracy or theresults to be obtained from the use of such information and nowarranty is expressed or implied concerning the use of theseproducts. The buyer assumes all risks of use and/or handling.Any user must make his own determination and satisfy himselfthat the products supplied by Lonza Group Ltd or its affiliatesand the information and recommendations given by LonzaGroup Ltd or its affiliates are (i) suitable for intended processor purpose, (ii) in compliance <strong>with</strong> environmental, health andsafety regulations, and (iii) will not infringe any third party’sintellectual property rights.© Copyright 2013, Lonza Walkersville, Inc. All rights reserved.XX-XX ABC 13/01GM-CA005
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