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Blue Cytokine Reporter Cells Blue Cytokine Reporter Cells is an expanding family of engineered cell lines designed to provide a simple, rapid and reliable method to monitor the activation of signaling pathways induced by key cytokines. They allow to detect these biologically active cytokines and can also be used to screen for compounds exhibiting agonist and antagonist activities. Blue Cytokine Reporter Cells include B16-Blue and HEK-Blue Cells. B16-Blue and HEK-Blue Cells are engineered murine melanoma and HEK293 cells, respectively. They express an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene that can be quantitatively detected using QUANTI-Blue , a SEAP colorimetric detection medium. Blue Cytokine Reporter Cells express this SEAP reporter gene under the control of specific promoters that are selectively activated by the cytokines or other immune modulators known to induce the pathway of interest. Therefore, in the presence of the cytokine, agonist or antagonist compound, the pathway is activated or inhibited modulating the SEAP activity. The amount of SEAP secreted in the cell supernatant can be measured spectrophotometrically with QUANTI-Blue . JAK/STAT Pathway Smad Pathway • B16-Blue IFN-a/b Cells • HEK-Blue TGF-b Cells • HEK-Blue IFN-a/b Cells • HEK-Blue IL-4/IL-13 Cells • HEK-Blue IL-6 Cells NF-kB Pathway CD40 Pathway • HEK-Blue TNF-a/IL-1b Cells • HEK-Blue CD40L Cells • HEK-Blue IL-1b Cells • HEK-Blue IL-18/IL-1b Cells • HEK-Blue IL-33/IL-1b Cells NEW CYTOKINE SENSOR CELLS IFNa IFNb IFNg IL-1b IL-4 IL-6 IL-13 IL-18 IL-33 TGF-b TNF-a CD40L INFO B16-Blue IFN-a/b cells +++ +++ - - - - - - - - - - p 106 HEK-Blue CD40L cells - - - +++ - - - - - - +++ ++++ p 115 HEK-Blue IFN-a/b cells +++ +++ - - - - - - - - - - p 107 HEK-Blue IL-1b cells - - - ++++ - - - - - - - - p 111 HEK-Blue IL-4/IL-13 cells +/- - - - ++ - ++ - - - - - p 108 HEK-Blue IL-6 cells - - - - - ++ - - - - - - p 109 HEK-Blue IL-18/IL-1b cells - - - +++ - - - ++++ - - - - p 112 HEK-Blue IL-33/IL-1b cells - - - +++ - - - - ++++ - - - p 113 HEK-Blue TGF-b cells - - - - - - - - - +++ - - p 114 HEK-Blue TNF-a/IL-1b cells - - - +++ - - - - - - ++++ - p 110 www.invivogen.com/cytokine-sensor-cells 105 CYTOKINE SIGNALING 4
CYTOKINE SIGNALING 4 B16-Blue IFN-a/b - Murine Type I IFNs Reporter Cells ➥ Monitor the JAK/STAT pathway ➥ Monitor the RIG-I/MDA-5 pathway ➥ Detection of murine IFN-a and IFN-b Background Interferon-alpha (IFN-a) and interferon beta (IFN-b), play an important role in viral infections. They bind to an IFN receptor complex consisting of two alpha chains (IFNAR1 and IFNAR2) and recruit JAK1 and TyK2. These kinases phosphorylate STAT1 and STAT2 leading to the formation of the ISGF3 complex. ISGF3 binds to IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG) to regulate their expression (figure 1). IFN-a and IFN-b are produced in response to viral pathogen associated molecular patterns (PAMPs), such as viral RNA and DNA. These PAMPs are recognized by cytoplasmic pattern recognition receptors including the RNA helicases RIG-I or MDA5. Stimulation of B16-Blue IFN-a/b cells with RIG-I/MDA-5 agonists, such as transfected poly(I:C), triggers the secretion of IFN-a and IFN-b. Description B16-Blue IFN-a/b cells allow the detection of bioactive murine type I IFNs by monitoring the activation of the JAK/STAT/ISGF3 pathway. They derive from the murine B16 melanoma cell line of C57B1/6 origin after stable transfection with a SEAP reporter gene under the control of the IFN-a/binducible ISG54 promoter. Stimulation of B16-Blue IFN-a/b cells with murine IFN-a or IFN-b, or type I IFN inducers, such as tranfected poly(I:C) or poly(dA:dT), activates the JAK/STAT/ISGF3 pathway and triggers the subsequent production of SEAP (figures 2&3). Levels of SEAP in the supernatant can be easily determined with QUANTI-Blue , a medium that turns purple/blue in the presence of SEAP and by reading the OD at 655 nm. • Detection range for mIFN-a: 10 2 - 10 4 IU/ml • Detection range for mIFN-b: 10 2 - 10 4 IU/ml B16-Blue IFN-a/b cells are resistant to Zeocin Contents and Storage B16-Blue IFN-a/b cells are grown in RPMI medium, 2 mM L-glutamine, 10% FBS supplemented with 100 μg/ml Zeocin . Each vial contains 5-7x 10 6 cells and is supplied with 10 mg Zeocin , 50 mg Normocin and 1 pouch of QUANTI-Blue . Cells are shipped on dry ice. PRODUCT QUANTITY CAT. CODE B16-Blue IFN-a/b cells 5-7 x 10 6 cells bb-ifnab Related Products Blasticidin, page 17 Anti-hIFN-a-IgA , page 104 Zeocin , page 18 QUANTI-Blue page 14 Poly(I:C) (HMW), page 77 Poly(I:C) (HMW)/LyoVec , page 80 Poly(dA:dT) Naked, page 80 Poly(dA:dT)/LyoVec, page 80 106 www.invivogen.com/cytokine-sensor-cells Figure 1: JAK/STAT and RIG-I/MDA-5 signaling pathways Figure 2: Stimulation of B16-Blue IFN-a/b cells by recombinant human and murine IFN-a and IFN-b was assessed by measuring the levels of SEAP using QUANTI-Blue . Figure 3: Response of B16-Blue IFN-a/b cells following their stimulation with naked poly(I:C) or poly(:C)/LyoVec. Induction of type I IFNs was assessed by determining the levels of SEAP using QUANTI-Blue .
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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
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Poly-Arginine-HA Tagged Reprogrammi
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LENTI-Smart - Lentiviral Vector Pr
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Lentiviral Vector Production and Ce
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Antibody generation using pFUSE-CHI
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Engineering Fc regions for altered
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pBROAD & pWHERE - Optimized Vector
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pSELECT-Tag - Expression of a GFP-,
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pVIVO - Expression of Two Genes of
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pORF Description pORF plasmids feat
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Prom A-List - Human and Rodent Pro
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PROMOTER GENE TISSUE DISTRIBUTION S
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ChemiComp E. coli ChemiComp E. coli
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Fast-Media ® Preparation 1. Empty
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RIG-I-like Receptors 54 RLR & RLR-R
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Toll-Like Receptors Toll-Like Recep
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www.invivogen.com/innate-immunity 5
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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 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 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
Page 122 and 123: Immunoglobulin A The mucosal surfac
Page 124 and 125: IgA Detection and Quantification In
Page 126 and 127: IgG Antibodies InvivoGen offers a s
Page 128 and 129: Mediators? 1. Leroux-Roels G., 2010
Page 130 and 131: Poly(I:C) Synthetic double-stranded
Page 132 and 133: pVAC - Induction of Neutralizing An
Page 134 and 135: CpG-Free DNA CpGs and the Immune Re
Page 136 and 137: pCpGfree-basic & pCpGfree-promoter
Page 138 and 139: pCpGfree-siRNA & pCpGfree-siRNADUO
Page 140 and 141: Custom-Made CpG-Free Genes InvivoGe
Page 143 and 144: RNA INTERFERENCE 7 psiRNA System I
Page 145 and 146: RNA INTERFERENCE 7 psiRNA-h7SK - 7S
Page 147 and 148: RNA INTERFERENCE 7 psiTEST System
Page 149 and 150: RNA INTERFERENCE 7 Ready-Made psiRN
Page 151 and 152: RNA INTERFERENCE 7 Custom-Made psiR
Page 153 and 154: INHIBITORS 8 Inhibitors PRODUCT DES
Page 155 and 156: 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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