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MEF Cells C3H/TLR4mut & C3H/WT MEFs TLR4 Reporter Murine Embryonic Fibroblasts C3H/TLR4mut and C3H/WT MEF cell lines were isolated from C3H/HeJ (TLR4-deficient) and C3H/HeN (wild-type) mouse embryos respectively. They stably express an NF-kB-inducible SEAP reporter construct that allows to monitor in a simple and convenient manner the activation of NF-kB. C3H/WT MEFs express high levels of TLR2 and TLR4 and low levels of TLR3 and TLR5. The presence of TLR2, TLR3, TLR4, or TLR5 agonists triggers a signaling cascade in C3H/WT MEFs leading to the activation of NF-kB and the subsequent induction of SEAP. The amount of SEAP secreted in the supernatant can be readily detected when using QUANTI-Blue , a SEAP detection medium. In C3H/TLR4mut MEFs, TLR4 agonists do not induce the activation of NF-kB and the production of SEAP in contrast to TLR2, TLR3 and TLR5 ligands. Thus these two cell lines provide a useful tool to determine whether a given compound is a specific TLR4 agonist. They can be used to assess the purity of a given LPS by detecting the presence of contaminants that stimulate TLR2 (see graph, LPS-EB standard versus LPS-EB ultrapure). Contents C3H/TLR4mut and C3H/WT MEF cell lines are grown in DMEM medium with 2mM L-glutamine, 10% FBS supplemented with 10 μg/ml blasticidin. Each vial contains 5-7 x 10 6 cells and is supplied with 1 mg blasticidin. Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free. C57/WT MEFs RLR Reporter Murine Embryonic Fibroblasts MEFs produce IFN-b in response to viral infection in a RLR-dependent manner1 . Thus, these cells are commonly used to study the RLR pathway. C57/WT MEFs were isolated from embryos under C57BL/6 background and immortalized with the SV40 large antigen. They stably express a SEAP reporter gene inducible by NF-kB and IRF3/7 providing a convenient method to monitor the activation of these transcription factors upon stimulation with a RIG-I/MDA-5 ligand. C57/WT MEFs express both RIG-I and MDA-5. Stimulation of C57/WT cells with poly(I:C)/LyoVec complexes induces the secretion of SEAP in a dose-dependent manner (see figure). In contrast, stimulation with naked poly(I:C) has no effect on SEAP secretion although the cells express also TLR3. These data confirm that C57/WT MEFs respond to viral dsRNA primarily through the RLR pathway1 . Both RIG-I and MDA-5 appear to respond to transfected poly(I:C) in MEFs2 . 1. Kato H. et al., 2005. Cell type-specific involvement of RIG-I in antiviral response. Immunity 23:19-28. 2. Venkataraman T. et al., 2007. Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. J Immunol. 178:6444-6455. Contents C57/WT MEFs are grown in DMEM medium with 10% FBS, 2 mM L-glutamine and supplemented with 100 μg/ml Zeocin and 3 μg/ml blasticidin. Each vial contains 5-7 x 10 6 cells and is supplied with 10 mg Zeocin and 1 mg blasticidin. Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free. www.invivogen.com/reporter-cells TLR stimulation profile of C3H/TLR4mut and C3H/WTMEF cells. Cells were incubated with TLR agonists: Pam3CSK4, 100 ng/ml (TLR2), poly(I:C), 10 μg/ml (TLR3), MPLAs, 10 μg/ml (TLR4), LPS-EB standard, 10 μg/ml (TLR4, TLR2), LPS-EB ultrapure, 10 μg/ml (TLR4), LPS-RS, 10 μg/ml (TLR2), flagellin, 1 μg/ml (TLR5). After 24h incubation, TLR stimulation was assessed by measuring the levels of SEAP in the supernatant by using QUANTI-Blue . PRODUCT QUANTITY CAT. CODE C3H/TLR4mut MEFs 5-7 x 10 6 cells mef-c3h4m C3H/WT MEFs 5-7 x 10 6 cells mef-c3hwt RLR stimulation in C57/WT MEFs. C57/MEFs were incubated with increasing concentrations of poly(I:C) or poly(I:C)/LyoVec complexes prepared extemporaneously at a 1:12 ratio. After 24h incubation, RLR stimulation was assessed by measuring the levels of SEAP secreted in the supernatant by using QUANTI- Blue , a SEAP detection medium. PRODUCT QUANTITY CAT. CODE C57/WT MEFs 5-7 x 10 6 cells mef-c57wt 71 INNATE IMMUNITY 3
INNATE IMMUNITY 3 Antibodies InvivoGen provides a collection of monoclonal and polyclonal antibodies targeting human and murine TLRs. Most of them have been developed in house. They are listed below and classified according to their application(s): ➥ Antibodies for neutralization. Some of them can be used also for flow cytometry ➥ Antibodies for detection. The applications include Western blotting, immunoprecipitation, immunohistochemistry and flow cytometry. • Anti-TLR-IgA antibodies are recombinant monoclonal IgA2 antibodies against TLRs. They have been developed by InvivoGen using proprietary techniques. They have been selected for their ability to efficiently block the biological activity of these TLRs. They can also be used for flow cytometry (FC). • Anti-TLR IgG antibodies are mouse monoclonal antibodies against TLRs. They have been generated by InvivoGen using DNA vaccination and screened for their ability to neutralize TLR activity. • MAb-TLR and Mab-Dectin-1 antibodies are monoclonal mouse IgG antibodies. They can be used for various applications but have been tested in our laboratories only for neutralization and flow cytometry. MAb-TLR antibodies are also available conjugated with FITC. • PAb-TLR antibodies are polyclonal antibodies against human extracellular TLRs, developed by InvivoGen. These antibodies have been generated by DNA vaccination in rats. They were obtained by purification of the IgG fraction from the sera by Protein G affinity chromatography. Antibodies for Neutralization 72 ANTIBODY DESCRIPTION SPECIFICITY APPLICATIONS QTY CAT. CODE Anti-CD14 Anti-hCD14-IgA Monoclonal human IgA2 Human CD14 Neutralization (TLR2, TLR4), FC 100 μg maba-hcd14 Anti-Dectin-1 MAb-mDectin-1 Monoclonal mouse IgG2b (clone 2A11) Mouse Dectin-1 Neutralization, FC 100 μg mab-mdect Anti-TLR1 Anti-hTLR1-IgG Monoclonal mouse IgG1 (H2G2) Human TLR1 Neutralization 100 μg mabg-htlr1 PAb-hTLR1 Polyclonal rat IgG Human TLR1 Neutralization 200 μg pab-hstlr1 Anti-TLR2 Anti-hTLR2-IgA Monoclonal human IgA2 Human TLR2 Neutralization, FC 100 μg maba2-htlr2 Anti-mTLR2-IgG Monoclonal mouse IgG2a (C9A12) Mouse TLR2 Neutralization 100 μg mabg-mtlr2 MAb-mTLR2 Monoclonal mouse IgG1 (T2.5) Human/mouse TLR2 Neutralization , FC, IHC 100 μg mab-mtlr2 PAb-hTLR2 Polyclonal rat IgG Human TLR2 Neutralization 200 μg pab-hstlr2 Anti-TLR4 Anti-hTLR4-IgA Monoclonal human IgA2 Human TLR4 Neutralization 100 μg maba2-htlr4 PAb-hTLR4 Polyclonal rat IgG Human TLR4 Neutralization 200 μg pab-hstlr4 Anti-TLR5 Anti-hTLR5-IgA Monoclonal human IgA2 Human TLR5 Neutralization, FC 100 μg maba2-htlr5 Anti-mTLR5-IgG Monoclonal rat IgG2a (Q23D11) Mouse TLR5 Neutralization 100 μg mabg-mtlr5 PAb-hTLR5 Polyclonal rat IgG Human TLR5 Neutralization 200 μg pab-hstlr5 Anti-TLR6 Anti-hTLR6-IgG Monoclonal mouse IgG1 (C5C8) Human TLR6 Neutralization 100 μg mabg-htlr6 PAb-hTLR6 Polyclonal rat IgG Human TLR6 Neutralization 200 μg pab-hstlr6 * FC: flowcytometry; IHC: immunohistochemistry; WB: Western blot www.invivogen.com/antibodies
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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
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Blasticidin S Description Blasticid
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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 68 and 69: Response of HEK-Blue hTLR2 cells t
Page 70 and 71: Ramos-Blue Cells - NF-kB/AP-1 Repo
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
Page 118 and 119: 5 ANTIBODIES & VACCINATION Antibodi
Page 120 and 121: 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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