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293/TLR & 293/NOD Clones 293/TLR Clones 293/TLR clones are HEK293 cells stably transfected with a pUNO-TLR or pDUO-TLR plasmid. They can be used to determine TLR activation upon ligand stimulation by assessing IL-8 production or NF-kB activation. The latter can be evaluated by transfecting transiently or stably 293/TLR clones with pNiFty, a family of NF-kB inducible reporter plasmids expressing either the secreted alkaline phosphatase or luciferase reporter gene (see page 74). 293/TLR-HA Clones 293/hTLR-HA clones were obtained by stably transfecting HEK293 cells with a pUNO-TLR-HA plasmid. pUNO-TLR-HA plasmids express TLR genes that have been fused at the 3’end to the influenza hemaglutinine (HA) tag. Addition of this tag has no deleterious effect on the expression and function of the TLR genes. The HA tag is the epitope of a very efficient and specific monoclonal antibody (see page 73). 293/NOD Clones 293/NOD clones are transfected HEK293 cells that express stably the NOD1 or NOD2 genes. These clones can be used to study NOD1 and NOD2 activation pathways following stimulation with their cognate ligand by assessing IL-8 production or NF-kB activation. NF-kB activation can be assessed using an NF-kB-inducible reporter system such as pNiFty. 293/Control Clones 293/Control clones were generated by stably transfecting HEK293 cells with a pUNO control plasmid expressing either the lacZ reporter gene (293/LacZ) or a multiple cloning site (293/null). Pam3CSK4 (100 ng/ml) Poly(I:C) (100 ng/ml) LPS-EB (100 ng/ml) Flagellin (10 ng/ml) Imiquimod (10 μg/ml) ssRNA40 (5 μg/ml) ODN 2006 (10 μg/ml) iEDAP (100 ng/ml) MDP (100 ng/ml) TNF-a (50 ng/ml) NI 293/null 293/hNOD1 293/hNOD2 293/hTLR2 293/hTLR3 293/hTLR4-MD2-CD14 293/hTLR5 293XL/null 293XL/hTLR7 293XL/hTLR8 293XL/hTLR9 TLR and NOD induction profile of 293 clones : 293/TLR, NOD and control clones were transfected transiently with pNiFty-SEAP and stimulated with TLR and NOD ligands. After 16 hour stimulation, NF-kB-induced SEAP activity was assessed using QUANTI-Blue , a SEAP-detection medium. Note: 293 clones and in particular 293XL clones express endogenous levels of TLR3 and TLR5, as well as TLR1, TLR6 and NOD1. CELL LINE 293/TLR Clones CAT. CODE (HUMAN) CAT. CODE (MOUSE) 293/MD2-CD14 293-hmd2cd14 - 293/TLR1 - 293-mtlr1 293/TLR1/2 - 293-mtlr1/2 293/TLR2 293-htlr2 293-mtlr2 293/TLR2-CD14 293-htlr2cd14 - 293/TLR2/6 293-htlr2/6 293-mtlr2/6 293/TLR3 293-htlr3 293-mtlr3 293/TLR4 293-htlr4a 293-mtlr4 293/TLR4-MD2-CD14 293-htlr4md2cd14 293-mtlr4md2cd14 293/TLR5 293-htlr5 293-mtlr5 293/TLR5-CD14 293-htlr5cd14 - 293/TLR6 - 293-mtlr6 293XL/TLR7* 293xl-htlr7 293xl-mtlr7 293XL/TLR8A* 293xl-htlr8 - 293/TLR9 - 293-mtlr9 293XL/TLR9A* 293xl-htlr9 - 293/hTLR-HA Clones 293/hTLR1-HA 293-htlr1ha - 293/hTLR2-HA 293-htlr2ha - 293/hTLR3-HA 293-htlr3ha - 293/hTLR4-HA 293-htlr4ha - 293/hTLR5-HA 293-htlr5ha - 293/hTLR6-HA 293-htlr6ha - 293XL/hTLR7-HA* 293xl-htlr7ha - 293XL/hTLR8-HA* 293xl-htlr8ha - 293XL/hTLR9-HA* 293xl-htlr9ha - 293/hTLR10-HA 293/NOD Clones 293-htlr10ha - 293/NOD1 293-hnod1 293-mnod1 293/NOD2 293-hnod2 293-mnod2 293/Control Clones 293/LacZ 293-lacz - 293/null 293-null - 293XL/null* 293xl-null - *293XL clones express the human anti-apoptotic Bcl-XL gene. Contents All 293 clones are grown in standard DMEM medium with 10% FBS, 2mM L-glutamine supplemented with blasticidin (10 μg/ml). Cells are provided frozen in a cryotube containing 5-7 x 10 6 cells and supplied with 100 μl of blasticidin at 10 mg/ml. Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free. Related Products www.invivogen.com/reporter-cells Blasticidin, page 17 TLR Ligands, pages 77-80 pNiFty, see page 74 NOD Ligands, see page 80 65 INNATE IMMUNITY 3
INNATE IMMUNITY 3 HEK-Blue TLR & HEK-Blue NOD Cells InvivoGen introduces HEK-Blue TLR and HEK-Blue NOD cells, a collection of engineered cell lines designed to provide a rapid, sensitive and reliable method to screen and validate TLR and NOD agonists or antagonists. They express an NF-kB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene that can be conveniently monitored using the SEAP detection media QUANTI-Blue or HEK-Blue Detection. HEK-Blue TLR cells HEK-Blue TLR cells are engineered HEK293 cells that stably co-express a human or murine TLR gene and an NF-kB-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. To increase the sensitivity to their cognate agonists, HEK-Blue TLR2 and HEK-Blue TLR4 cells were further transfected with the co-receptors CD14 and MD2/CD14, respectively. HEK-Blue TLR cells are resistant to the selective antibiotics blasticidin and Zeocin . HEK-Blue TLR2 and HEK-Blue TLR4 cells are additionally resistant to hygromycin. HEK-Blue NOD cells HEK-Blue NOD cells are engineered HEK293 cells that stably co-express the human and murine NOD1 or NOD2 gene and an NF-kB-inducible SEAP reporter gene. HEK-Blue NOD cells are resistant to blasticidin and Zeocin . HEK-Blue Null cells HEK-Blue Null cells are the parental cell lines used to generate HEK-Blue TLR and HEK-Blue NOD cells. • HEK-Blue Null1 cells express the SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB binding sites.This cell line is the parental cell line of HEK-Blue hTLR2, hTLR3, hTLR5, hTLR8, h/mTLR9 and h/mNOD1 cells. • HEK-Blue Null1-k cells express the same reporter system than HEK- Blue Null1 cells but are slightly different genotypically. This cell line is the parental cell line of HEK-Blue mTLR3 and hTLR7 cells. • HEK-Blue Null1-v cells express the same reporter system than HEK- Blue Null1 cells but are slightly different genotypically. This cell line is the parental cell line of HEK-Blue mTLR4 and mTLR8 cells. • HEK-Blue Null2 cells express the SEAP reporter gene under the control of the IL-12 p40 minimal promoter fused to five NF-kB binding sites.This cell line is the parental cell line of HEK-Blue mTLR2, hTLR4 and h/mNOD2 cells. • HEK-Blue Null2-k cells express the same reporter system than HEK- Blue Null2 cells but are slightly different genotypically. This cell line is the parental cell line of HEK-Blue mTLR5 and mTLR7 cells. HEK-Blue Null cells are resistant to Zeocin . Principle Recognition of a TLR or NOD agonist by its cognate receptor triggers a signaling cascade leading to the activation of NF-kB and the production of SEAP (figure 1). SEAP levels can be determined spectrophotometrically using HEK-Blue Detection or QUANTI-Blue , both are SEAP detection media that turn purple/blue in the presence of alkaline phosphatase. Quality Control Expression of exogenous TLRs, NOD,1/2, CD14 and/or MD2 was confirmed by RT-PCR. The functionality of each cell line is validated through stimulation assays performed with a selection of TLR and NOD agonists. Note: HEK-Blue TLR, HEK-Blue NOD and HEK-Blue Null cells express endogenous levels of TLR3, TLR5 and NOD1. 66 PRODUCT www.invivogen.com/reporter-cells TLR- and NOD-induced NF-kB signaling pathway CAT. CODE (human) CAT. CODE (mouse) HEK-Blue MD2-CD14 Cells hkb-hmdcd NEW - HEK-Blue TLR2 Cells hkb-htlr2 hkb-mtlr2 NEW HEK-Blue TLR3 Cells hkb-htlr3 hkb-mtlr3 NEW HEK-Blue TLR4 Cells hkb-htlr4 hkb-mtlr4 NEW HEK-Blue TLR5 Cells hkb-htlr5 hkb-mtlr5 NEW HEK-Blue TLR7 Cells hkb-htlr7 hkb-mtlr7 NEW HEK-Blue TLR8 Cells hkb-htlr8 hkb-mtlr8 NEW HEK-Blue TLR9 Cells hkb-htlr9 hkb-mtlr9 NEW HEK-Blue Null1 Cells hkb-nul11 - HEK-Blue Null1-k Cells hkb-nul11k - HEK-Blue Null1-v Cells hkb-nul11v - HEK-Blue Null2 Cells hkb-nul12 - HEK-Blue HEK-Blue Null2-k Cells hkb-nul12k - NOD1 Cells hkb-hnodl hkb-mnodl NEW HEK-Blue HEK-Blue NOD2 Cells hkb-hnod2 hkb-mnod2 NEW TLR Cells HEK-Blue NOD Cells HEK-Blue Null Cells Contents and Storage HEK-Blue TLR, HEK-Blue NOD and HEK-Blue Null cells are grown in DMEM medium, 2mM L-glutamine, 10% FBS and supplemented with 100 μg/ml Zeocin , 30 μg/ml blasticidin and/or 200 μg/ml HygroGold (ultra-pure hygromycin) depending on the cell line. Cells are provided frozen in a cryotube containing 5-7 x 10 6 cells and supplied with the corresponding selective antibiotic(s), 1 ml Normocin (50 mg/ml) and 1 pouch of QUANTI-Blue . Cells are shipped on dry ice.
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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
Page 16 and 17: 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 68 and 69: Response of HEK-Blue hTLR2 cells t
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
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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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