4 CYTOKINE SIGNALING Cytokine Genes 104 Cytokine Antibodies 104 Cytokine Reporter Cells 105-116 Cytokine and Related Genes Cytokines are key modulators of the immune system. They are produced mainly by T lymphocytes in response to infectious agents. Some cytokines induce the inflammatory response or stimulate B cells to produce antibodies while others act to offset these effects by downregulation. InvivoGen offers a comprehensive list of cytokine genes classified in different families including interleukins and chemokines. Description Cytokine and related genes are provided either in a pORF or a pUNO plasmid (see page 37), both containing the complete coding sequence from the ATG to the Stop codon. Each gene is fully sequenced. Gene families in pORF plasmids: - Chemokine genes - Immune receptor genes - Chemokine receptor genes - Interferon genes - Cytokine genes - Interleukin genes - Cytokine suppressor genes - Interleukin receptor genes Gene family in pUNO plasmids: - Interferon signaling genes Contents and Storage Each pORF and pUNO plasmid is provided as a lyophilized transformed PRODUCT QUANTITY CAT. CODE* E. coli strain on a paper disk. Transformed strains are shipped at room temperature and should be stored at -20°C. pORF-<gene> E. coli disk porf-<gene> Each pORF is provided with 4 pouches of E. coli Fast-Media® Amp (2 TB pUNO-<gene> E. coli disk puno-<gene> and 2 Agar) and each pUNO plasmid with 4 pouches of E. coli Fast-Media® Blas (see pages 44-45). * Catalog codes are available on our website Anti-Cytokine Neutralizing IgA Antibodies 4 Description Contents and Storage Neutralizing IgA antibodies are chimeric monoclonal antibodies in which Each neutralizing IgA antibody is provided lyophilized from a 0.2 μm the constant domains of the human IgA molecule were combined with filtered solution in PBS. Product should be reconstituted in sterile water. murine variable regions. They have been selected for their ability to Lyophilized antibodies are stable greater than six months when stored efficiently neutralize the biological activity of selected cytokines. The at -20ºC. Reconstituted IgAs are stable 1 month when stored at 4ºC and neutralizing activity of these IgA antibodies was determined using 6 months when aliquoted and stored at -20ºC. InvivoGen’s HEK-Blue™ Cytokine Cells. CATALOG PRODUCT ANTIGEN REACTIVITY QUANTITY CODE Anti-hCD40L-IgA2 Human CD40 ligand Human 100 μg maba-h40l Anti-hIFNa-IgA2 Human Interferon a Human 100 μg maba-hifna Anti-hIFNg-IgA2 Human Interferon g Human 100 μg maba-hifng Anti-hIL-1b-IgA2 Human Interleukin 1b Human 100 μg maba-hil1b CYTOKINE SIGNALING Anti-hIL-4-IgA2 Human Interleukin 4 Human 100 μg maba-hil4 Anti-hIL-6-IgA2 Human Interleukin 6 Human 100 μg maba-hil6 Anti-hIL-13-IgA2 Human Interleukin 13 Human 100 μg maba-hil13 Anti-hIL-18-IgA2 Human Interleukin 18 Human 100 μg maba-hil18 Anti-hTGFb-IgA2 Human Tumor Growth Factor b Human 100 μg maba-htgfb Anti-hTNFa-IgA2 Human Tumor Necrosis Factor a Human 100 μg htnfa-mab7 104 www.invivogen.com/cytokine-signaling www.invivogen.com/cytokine-sensor-cells 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 4 • 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 CYTOKINE SIGNALING 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 ™ 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 Figure 1: JAK/STAT and RIG-I/MDA-5 signaling pathways 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/b- inducible 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: 102 - 104 IU/ml • Detection range for mIFN-b: 102 - 104 IU/ml 4 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, Figure 2: Stimulation of B16-Blue™ IFN- / cells by recombinant human and 10% FBS supplemented with 100 μg/ml Zeocin™. Each vial contains a b 6 murine IFN-a and IFN-b was assessed by measuring the levels of SEAP using 5-7x 10 cells and is supplied with 10 mg Zeocin™, 50 mg Normocin™ and QUANTI-Blue™. 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice. PRODUCT QUANTITY CAT. CODE B16-Blue™ IFN-a/b cells 5-7 x 106 cells bb-ifnab CYTOKINE SIGNALING 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 Figure 3: Response of B16-Blue™ IFN-a/b cells following their stimulation with Poly(dA:dT) Naked, page 80 Poly(dA:dT)/LyoVec, page 80 naked poly(I:C) or poly(:C)/LyoVec. Induction of type I IFNs was assessed by determining the levels of SEAP using QUANTI-Blue™. 106 www.invivogen.com/cytokine-sensor-cells www.invivogen.com/cytokine-sensor-cells HEK-Blue™ IFN-a/b Cells - Human Type I IFN Reporter Cells ➥ Monitor the JAK/STAT/ISGF3 pathway ➥ Detection of human IFN-a and IFN-b Background Type I interferons, in particular interferon alpha (IFN-a) and interferon beta (IFN-b), play a vital role in host resistance to viral infections. They signal mainly through the JAK-STAT pathway. Following their production, IFN-a and IFN-b bind to a common receptor (IFNAR) and recruit the Janus kinases (JAK1 and TyK2). JAKs phosphorylate STAT1 and STAT2, which then dimerize and interact with IFN regulatory factor 9 (IRF9), forming a complex named ISGF3. ISGF3 binds to IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG) to regulate their expression (figure 1). Description HEK-Blue™ IFN-a/b cells allow the detection of bioactive human type I IFNs by monitoring the activation of the ISGF3 pathway. These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. The other genes of the pathway (IFNAR1, IFNAR2, JAK1, TyK2 and STAT1) are naturally expressed in sufficient amounts. The cells were further transfected with a SEAP reporter gene under the control of the IFN- / -inducible a b Figure 1: JAK-STAT pathway induced by type I IFNs.