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Response of HEK-Blue hTLR2 cells to TLR2 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 0.1 ng/ml FSL-1 (TLR2/6), 0.1 ng/ml Pam3CSK4(TLR1/2), 10 7 cells/ml HKLM, 1 μg/ml LTA- SA, 1 ng/ml LPS-PG or 1 μg/ml PGN-BS. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. Response of HEK-Blue hTLR5 cells to TLR5 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 100 ng/ml FLA-BS, 10 ng/ml FLA-ST ultrapure or 10 ng/ml recFLA-ST. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. Response of HEK-Blue hTLR9 cells to TLR9 agonists. Cells were stimulated with 1 μg/ml ODN2006 (type B), 1 μg/ml ODN2216 (type A) or 5 μg/ml E. coli ssDNA. After 24h incubation, the levels of NF-kB-induced SEAP were determined using QUANTI-Blue by reading the OD at 655 nm. Response of HEK-Blue hTLR3 cells to TLR3 agonists. Cells were stimulated with 100 ng/ml poly(I:C) (HMW), 100 ng/ml poly(I:C)-LMW or 1 μg/ml poly(A:U). After 24h incubation, the levels of NF-kBinduced SEAP were determined using QUANTI-Blue by reading the OD at 655 nm. Response of HEK-Blue hTLR7 cells to TLR7/8 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 100 ng/ml CL264, 5 μg/ml imiquimod, 1 μg/ml gardiquimod or 100 ng/ml R848. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. Response of HEK-Blue hNOD1 cells to NOD1/2 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 1 μg/ml iE-DAP, 100 ng/ml C12-iE-DAP, 1 μg/ml TRI-DAP or 1 μg/ml M-TriDAP. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. www.invivogen.com/reporter-cells Response of HEK-Blue hTLR4 cells to TLR4 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 10 ng/ml LPS-EB ultrapure, 10 ng/ml LPS-EK ultrapure, 100 ng/ml MPLA or 100 ng/ml MPLAs. After 24h incubation, the levels of NF-kBinduced SEAP were determined by reading the OD at 655 nm. Response of HEK-Blue hTLR8 cells to TLR7/8 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 5 μg/ml ssRNA40, 1 μg/ml CL075 or1 μg/ml R848. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. Response of HEK-Blue hNOD2 cells to NOD1/2 agonists. Cells were incubated in HEK-Blue Detection medium and stimulated with 100 ng/ml MDP, 10 ng/ml L18-MDP 100 ng/ml murabutide or 100 ng/ml M-TriDAP. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm. 67 INNATE IMMUNITY 3
INNATE IMMUNITY 3 THP1-XBlue Cells - NF-kB/AP-1 Reporter Monocytes THP1-XBlue are derived from THP-1, a human immune cell line that naturally expresses most TLRs. They stably express an NF-κB/AP-1-inducible reporter (SEAP) system to facilitate the monitoring of TLR-induced NF-kB/AP-1 activation. Three cell lines are available: THP1-XBlue , THP1-XBlue -CD14, which overexpresses the cell surface protein CD14 for enhanced sensitivity, and THP1-XBlue -defMyD characterized by a deficient MyD88 activity. THP1-XBlue THP1-XBlue cells were obtained by stable transfection of THP-1 cells with a reporter construct expressing a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-kB and AP-1. Upon TLR stimulation, THP1-XBlue cells induce the activation of NF-kB and AP-1 and subsequently the secretion of SEAP. The reporter protein is easily detectable and measurable when using QUANTI-Blue , a medium that turns purple/blue in the presence of SEAP (see page 14). THP1-XBlue cells are resistant to the selectable marker Zeocin . THP1-XBlue -MD2-CD14 NEW THP1-XBlue -MD2-CD14 cells derive from the THP1-XBlue cell line by cotranfection of the MD2 and CD14 genes. MD2 is an accessory molecule essential for LPS-induced TLR4 response. CD14 interacts with several TLRs, including TLR4 and TLR2. Its overexpression was found to increase the reponse to the majority of TLR ligands (Figure 1). THP1-XBlue -CD14 cells are resistant to the antibiotics Zeocin and G418. THP1-XBlue -defMyD THP1-XBlue -defMyD cells are THP1-XBlue cells deficient in MyD88 activity. They are unable to respond to the activation of receptors which signaling is dependent on MyD88, such as TLR2, TLR4 and IL-1Rs. They remain responsive to MyD88-independent receptors, such as NOD1 and TNFR (Figure 2). Quality Control Expression of the TLR (TLR1 to TLR10), MD2 and CD14 genes was determined by RT-PCR in THP1-XBlue , THP1-XBlue-MD2-CD14 and THP1-XBlue-defMyD cells. All TLR mRNAs were detected but the cells do not respond to all TLR agonists (Figure 1). Considering the concentration of ligands used to stimulate these cells, TLR2, TLR2/1and TLR2/6 responses are considered to be very strong. TLR4, TLR5, TLR8, NOD1 and NOD2 responses are robust. TLR7 response is weak (a very high concentration of its cognate ligands is needed to detect a response). TLR3 and TLR9 responses are not detectable even when high concentrations of the ligands are used. MyD88 deficiency in THP1-XBlue-defMyD cells was confirmed by qRT-PCR. Contents THP1-XBlue , THP1-XBlue-MD2-CD14 and THP1-XBlue-defMyD cells are grown in RPMI medium, 2mM L-glutamine, 10% FBS supplemented with the appropriate selective antibiotic(s): 200 μg/ml Zeocin for THP1-XBlue ; 200 μg/ml Zeocin and 250 μg/ml G418 for THP1-XBlue-MD2-CD14; 200 μg/ml Zeocin and 100 μg/ml HygroGold for THP1-XBlue-defMyD. Cells are provided in a vial containing 5-7 x 10 6 cells and is supplied with 10 mg Zeocin (and 10 mg G418 or 10 mg HygroGold ) and 1 pouch of QUANTI-Blue . Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free. 68 www.invivogen.com/reporter-cells Figure 1: TLR and NOD stimulation profile in THP1-XBlue and THP1-XBlue - MD2-CD14. Cells were incubated with 1 ng/ml Pam3CSK4(TLR1/2), 10 ng/ml FSL- 1 (TLR2/6), 10 7 cells/ml HKLM (TLR2), 10 μg/ml poly(I:C) (TLR3), 10 ng/ml LPS-EK (TLR4), 10 ng/ml RecFLA-ST (TLR5), 5 μg/ml imiquimod (TLR7), 5 μg/ml CL075 (TLR8), 10 μg/ml ODN2006 (TLR9), 100 ng/ml C12-iEDAP (NOD1) or 10 μg/ml MDP (NOD2). After 24h incubation, TLR/NOD stimulation was assessed by measuring the levels of SEAP in the supernatant by using QUANTI-Blue . Figure 2: MyD88-dependent and -independent responses in THP1-XBlue and THP1-XBlue -defMyD. Cells were incubated with 1 μg/ml FSL-1 (TLR2/6), 1 μg/ml LPS-EK (TLR4), 5 μg/ml CL075 (TLR8), 10 μg/mlTri-DAP (NOD1) and 50 ng/ml TNF-a. After 24h incubation, NF-kB activation was determined using QUANTI-Blue . PRODUCT QUANTITY CAT. CODE THP1-XBlue Cells 5-7 x 10 6 cells thpx-sp THP1-XBlue -MD2-CD14 Cells 5-7 x 10 6 cells thpx-mdcdsp THP1-XBlue -defMyD 5-7 x 10 6 cells thpx-dmyd
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INNOVATION WITHIN REACH InvivoGen
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CONTENTS AT A GLANCE 1 CELL CULTURE
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4 CYTOKINE SIGNALING TABLE of CONTE
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Mycoplasma Detection & Elimination
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PlasmoTest Contents - HEK-Blue -2
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Primocin - The Antimicrobial Shiel
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SEAP Reporter Gene System Secreted
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HEK-Blue Detection - Real-Time Det
Page 18 and 19: Blasticidin S Description Blasticid
Page 20 and 21: LyoVec New formulation LyoVec is
Page 22 and 23: Poly-Arginine-HA Tagged Reprogrammi
Page 24 and 25: LENTI-Smart - Lentiviral Vector Pr
Page 26 and 27: Lentiviral Vector Production and Ce
Page 28 and 29: Antibody generation using pFUSE-CHI
Page 30 and 31: Engineering Fc regions for altered
Page 32 and 33: pBROAD & pWHERE - Optimized Vector
Page 34 and 35: pSELECT-Tag - Expression of a GFP-,
Page 36 and 37: pVIVO - Expression of Two Genes of
Page 38 and 39: pORF Description pORF plasmids feat
Page 40 and 41: Prom A-List - Human and Rodent Pro
Page 42 and 43: PROMOTER GENE TISSUE DISTRIBUTION S
Page 44 and 45: ChemiComp E. coli ChemiComp E. coli
Page 46 and 47: Fast-Media ® Preparation 1. Empty
Page 48 and 49: RIG-I-like Receptors 54 RLR & RLR-R
Page 50 and 51: Toll-Like Receptors Toll-Like Recep
Page 52 and 53: www.invivogen.com/innate-immunity 5
Page 54 and 55: IPAF and NAIP5 IPAF (NLRC4, CLAN/CA
Page 56 and 57: C-Type Lectin Receptors C-type lect
Page 58 and 59: pUNO pUNO-HA Toll-Like Receptors (T
Page 60 and 61: GENE NAME/ALIASES DESCRIPTION CAT.
Page 62 and 63: pDUO - Gene Associations Features a
Page 64 and 65: pZERO-TLR & pDeNy - Dominant Negati
Page 66 and 67: 293/TLR & 293/NOD Clones 293/TLR Cl
Page 70 and 71: Ramos-Blue Cells - NF-kB/AP-1 Repo
Page 72 and 73: MEF Cells C3H/TLR4mut & C3H/WT MEFs
Page 74 and 75: Antibodies for Detection ANTIBODY D
Page 76 and 77: - NF-kB binding site: Nuclear facto
Page 78 and 79: PRODUCT ORIGIN/DESCRIPTION TLR2 Ago
Page 80 and 81: PRODUCT ORIGIN/DESCRIPTION TLR9 Ago
Page 82 and 83: PRODUCT ORIGIN/DESCRIPTION Dectin-1
Page 84 and 85: LPS-PG (Porphyromonas gingivalis) R
Page 86 and 87: • FLA-ST ultrapure was purified b
Page 88 and 89: 1. Zhao H. et al., 2004. Contributi
Page 90 and 91: 1. Hornung V. et al., 2006. 5'-Trip
Page 92 and 93: TLR/NOD Ligand Screening InvivoGen
Page 94 and 95: Signal Transduction Inhibitors Invi
Page 96 and 97: Pepinh-MYD - MyD88 inhibitory pepti
Page 98 and 99: 1. Martinon F,. et al., 2002. The i
Page 100 and 101: Nigericin - NLRP3 Inflammasome Indu
Page 102 and 103: Autophagy is a pathway by which cyt
Page 104 and 105: 4 CYTOKINE SIGNALING Cytokine Genes
Page 106 and 107: Blue Cytokine Reporter Cells Blue
Page 108 and 109: HEK-Blue IFN-a/b Cells - Human Typ
Page 110 and 111: HEK-Blue IL-6 Cells - IL6 Reporter
Page 112 and 113: HEK-Blue IL-1b Cells - IL-1b Repor
Page 114 and 115: HEK-Blue IL-33/IL-1b Cells - IL-33
Page 116 and 117: HEK-Blue CD40L Cells - CD40L Repor
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5 ANTIBODIES & VACCINATION Antibodi
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Antibody generation using pFUSE-CHI
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Immunoglobulin A The mucosal surfac
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IgA Detection and Quantification In
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IgG Antibodies InvivoGen offers a s
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Mediators? 1. Leroux-Roels G., 2010
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Poly(I:C) Synthetic double-stranded
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pVAC - Induction of Neutralizing An
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CpG-Free DNA CpGs and the Immune Re
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pCpGfree-basic & pCpGfree-promoter
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pCpGfree-siRNA & pCpGfree-siRNADUO
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Custom-Made CpG-Free Genes InvivoGe
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RNA INTERFERENCE 7 psiRNA System I
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RNA INTERFERENCE 7 psiRNA-h7SK - 7S
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RNA INTERFERENCE 7 psiTEST System
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RNA INTERFERENCE 7 Ready-Made psiRN
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RNA INTERFERENCE 7 Custom-Made psiR
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INHIBITORS 8 Inhibitors PRODUCT DES
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INHIBITORS 8 Pattern Recognition Re
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INHIBITORS 8 Triptolide - NF-kB Inh
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INHIBITORS 8 Heat Shock Protein Inh
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9 CUSTOM SERVICES TLR/NOD Ligand Sc
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CUSTOM SERVICES 9 Custom Cloning :
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CUSTOM SERVICES 9 Prices 164 TERMS
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166 PRODUCT (QUANTITY) CAT. CODE PA
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A 17-AAG 17-AAGH2 17-AEP-GA 2-Amino
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- ODN 4084-F - ODN INH-1 - ODN INH-
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ARGENTINA Genbiotech S.R.L. Tel: +5
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