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-
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. ISG54 promoter. Stimulation of HEK-Blue™ IFN-a/b cells with human IFN-a or IFN-b activates the JAK/STAT/ISGF3 pathway and subsequently induces the production of SEAP. 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 (figure 2). Stimulation of HEK-Blue™ IFN-a/b cells with recombinant human IFN-a can be blocked by anti-hIFN-a-IgA, a neutralizing monoclonal antibody of the IgA isotype (see page 104).
• Detection range for hIFN-a: 5 - 104 IU/ml • Detection range for hIFN-b: 20 - 104 IU/ml HEK-Blue™ IFN-a/b cells are resistant to blasticidin and Zeocin™. 4 Contents and Storage
HEK-Blue™ IFN-a/b cells are grown in standard DMEM medium, 2mM L-glutamine,10% FBS supplemented with blasticidin (30 μg/ml) and Zeocin™ (100 μg/ml). Cells are provided frozen in a cryotube containing 5-7 x 106 cells and supplied with 100 μl of blasticidin at 10 mg/ml, 100 μl Figure 2: Stimulation of HEK-Blue™ IFN- / cells by human IFN- 2b, IFN- 1a and of Zeocin™ at 100 mg/ml, 1 ml Normocin™ at 50 mg/ml, and 1 pouch of a b a b IFN-g was assessed by measuring the levels of SEAP using QUANTI-Blue™. QUANTI-Blue™. Cells are shipped on dry ice.
PRODUCT QUANTITY CAT. CODE Related Products HEK-Blue™ IFN-a/b cells 5-7x 106 cells hkb-ifnab Blasticidin, page 17 Anti-hIFN-a-IgA , page 104 ™ ™
Zeocin , page 18 QUANTI-Blue page 14 CYTOKINE SIGNALING
www.invivogen.com/cytokine-sensor-cells 107 HEK-Blue™ IL-4/IL-13 Cells - IL-4 and IL-13 Reporter Cells
➥ Monitor the JAK/STAT-6 pathway ➥ Detection of human IL-4 and human/murine IL-13
Background
The transcription factor STAT6 is activated primarily by two cytokines with overlapping biologic functions, IL-4 and IL-13. It can also be activated by IFN-a in a cell-specific manner. In non-hematopoietic cells, IL-4 and IL-13 bind a receptor complex composed of the IL-4Ralpha and IL-13Ralpha1. Upon ligand binding, the receptor complex activates the receptor-associated Janus kinases (JAK1 and Tyk2) leading to the recruitment of STAT6 and its phosphorylation. Activated STAT6 forms homodimers that translocate to the nucleus where they bind the promoter of responsive genes inducing gene transcription.
Description
HEK-Blue™ IL-4/IL-13 cells allow the detection of bioactive IL-4 and IL-13 by monitoring the activation of the STAT-6 pathway. These cells were generated by stable transfection of HEK293 cells with the human STAT6 gene to obtain a fully active STAT6 pathway. The other genes of the pathway are naturally expressed in sufficient amounts. The cells were further transfected with a SEAP reporter gene under the control of the IFNb minimal promoter fused to four STAT6 binding sites. HEK-Blue™ Figure 1: JAK/STAT6 signaling pathway induced by IL-4 and IL-4/IL-13 cells produce SEAP in response to IL-4 or IL-13 stimulation IL-13. and to a lower extent IFN-a. The levels of SEAP secreted in the supernatant can be easily determined with QUANTI-Blue™, a SEAP detection medium (Figure 2). Stimulation of HEK-Blue™ IL-4/IL-13 cells with IL-4 or IL-13 can be blocked by the neutralizing monoclonal anti-hIL-4-IgA and anti-hIL-13-IgA antibodies, respectively (see page 104).
• Detection range for IL-4: 0.5 - 100 ng/ml • Detection range for IL-13: 5 - 1000 ng/ml
HEK-Blue™ IL-4/IL-13 cells are resistant to blasticidin and Zeocin™.
4 Contents and Storage
HEK-Blue™ IL-4/IL-13 cells are grown in standard DMEM medium, 2 mM L-glutamine with 10% FBS supplemented with blasticidin (10 μg/ml) and Zeocin™ (100 μg/ml). Each vial contains 5-7 x 106 cells and is supplied with 100 μl of blasticidin at 10 mg/ml, 100 μl of Zeocin™ at 100 mg/ml, 1 ml Normocin™ at 50 mg/ml and 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice. Figure 2: Stimulation of HEK-Blue™ IL-4/IL-13 cells by recombinant human IL-4 and IL-13 was assessed by measuring the levels of SEAP secreted in the supernatant using HEK-Blue™ IL-4/IL-13 cells are also available in a kit. See below: QUANTI-Blue™.
HEK-Blue™ IL-4/IL-13 Kit
• HEK-Blue™ IL-4/IL-13 Cells 5-7 x 106 cells
CYTOKINE SIGNALING • Anti-hIL-4-IgA antibody 100 μg • Anti-hIL-13-IgA antibody 100 μg PRODUCT QUANTITY CAT. CODE ™ • QUANTI-Blue 1 pouch (100 ml) HEK-Blue™ IL-4/IL-13 Cells 5-7 x 106 cells hkb-stat6 • Normocin™ 1 ml (50 mg/ml) HEK-Blue™ IL-4/IL-13 Kit 1 kit hkb-stat6-kit • Blasticidin 100 μl (10 mg/ml) • Zeocin™ 100 μl (100 mg/ml)
108 www.invivogen.com/cytokine-sensor-cells www.invivogen.com/cytokine-sensor-cells HEK-Blue™ IL-6 Cells - IL6 Reporter Cells
➥ Monitor the JAK/STAT-3 pathway ➥ Detection of human IL-6
Background
The proinflammatory cytokine IL-6 is one of the most important mediators of fever and of the acute phase response. IL-6 exerts its action by first binding to the IL-6R. The complex of IL-6 and IL-6R associates with the signal-transducing membrane protein gp130, thereby inducing its dimerization. This leads to the activation by phosphorylation of the tyrosine kinases of the Janus family (JAK1, JAK2, and Tyk2). Activated JAKs induce the dimerization and translocation to the nucleus of STAT3 where it binds enhancer elements of IL-6-inducible genes.
Description
HEK-Blue™ IL-6 cells allow the detection of bioactive IL-6 by monitoring the activation of the STAT-3 pathway. These cells were generated by stable transfection of HEK293 cells with the human IL-6R gene. They were further transfected with a SEAP reporter gene under the control of the IFN-b minimal promoter fused to four STAT3 binding sites. Upon IL-6 stimulation, HEK-Blue™ IL-6 cells trigger the activation of STAT3 and the subsequent secretion of SEAP. SEAP can be readily monitored when using the SEAP detection medium Figure 1: JAK/STAT3 signaling pathway. QUANTI-Blue™. Stimulation of HEK-Blue™ IL-6 cells with recombinant human IL-6 can be blocked by anti-hIL-6-IgA, a neutralizing monoclonal antibody of the IgA isotype (see page 104).
• Detection range for IL-6: 0.5 - 50 ng/ml
HEK-Blue™/IL-6 cells are resistant to both hygromycin and Zeocin™.
Contents and Storage
HEK-Blue™ IL-6 cells are grown in standard DMEM medium, 2 mM L-glutamine, 10% FBS supplemented with 200 µg/ml HygroGold (ultrapure hygromycin) and 100 µg/ml Zeocin™. Cells are provided frozen 4 in a cryotube containing 5-7 x 106 cells and supplied with 100 µl of HygroGold™ at 100 mg/ml, 100 µl of Zeocin™ at 100 mg/ml, 1 ml Normocin™ at 50 mg/ml and 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice.
HEK-Blue™ IL-6 cells are also available in a kit. See below:
Figure 2: Stimulation of HEK-Blue™ IL-6 cells by recombinant human IL-6 was assessed by measuring the levels of SEAP using QUANTI-Blue™.
HEK-Blue™ IL-6 Kit
• HEK-Blue™ IL-6 Cells 5-7 x 106 cells
• Anti-hIL-6-IgA antibody 100 μg CYTOKINE SIGNALING • QUANTI-Blue™ 1 pouch (100 ml) PRODUCT QUANTITY CAT. CODE ™ • Normocin 1 ml (50 mg/ml) HEK-Blue™ IL-6 Cells 5-7 x 106 cells hkb-il6 • HygroGold™ 100 μl (100 mg/ml) HEK-Blue™ IL-6 Kit 1 kit hkb-il6-kit • Zeocin™ 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells 109 HEK-Blue™ TNF-a/IL-1b Cells - TNF-a and IL-1b Reporter Cells
➥ Monitor the TNF-a- and IL-1b-induced NF-kB and AP-1 pathways ➥ Detection of human and murine TNF-a and IL-1b(a)
Background
Tumor necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1b) are proinflammatory cytokines produced in response to microbial infection. The common inflammatory responses of TNF-a and IL-1b are mediated by interactions of these cytokines with distinct receptors: TNF-a binds TNFR1 and TNFR2, IL-1b binds IL-1RI. Binding of these cytokines to their receptors induces intracellular signaling. TNF-a signaling involves TRADD, TRAF2 and RIP, while IL-1b signaling is mediated by MyD88, IRAK-1, TRAF-6, and Rac1. Both signalings lead to the activation of the transcription factors NF-kB and AP-1 (figure 1).
Description
HEK-Blue™ TNF-a/IL-1b cells are designed to detect bioactive TNF-a and IL-1b (and IL-1a) by monitoring the activation of the NF-kB pathway. HEK-Blue™ TNF-a/IL-1b cells derive from HEK293 cells. They endogenously express the receptors for TNF-a and IL-1b and stably express a SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB and five AP-1 binding sites. HEK-Blue™ TNF-a/IL-1b cells secrete SEAP upon stimulation by human Figure 1: NF-kB/AP-1 signaling pathways induced by TNF-a and IL-1b. TNF-a and IL-1b (figure 2). SEAP production is also detected in the presence of murine TNF-a and IL-1b, and human or murine IL-1a. SEAP levels can be readily determined using a SEAP detection medium, such as QUANTI-Blue™ or HEK-Blue™ Detection medium. Stimulation of HEK-Blue™ TNF-a/IL-1b cells with recombinant human TNF-a or IL-1b can be blocked by the neutralizing monoclonal anti- hTNF-a-IgA or anti-hIL-1b-IgA antibody, respectively (see page 104).
• Detection range for TNF-a: 0.5 ng - 1 μg/ml • Detection range for IL-1b: 0.2 - 100 ng/ml 4 HEK-Blue™ TNF-a/IL-1b cells are resistant to Zeocin™. Contents and Storage
HEK-Blue™ TNF-a/IL-1b cells are grown in DMEM medium, 2 mM L-glutamine, 10% FBS supplemented with Zeocin™ (100 μg/ml). Each vial contains 5-7 x 106 cells and supplied with 100 μl of Zeocin™ at 100 mg/ml, 1 ml Normocin™ at 50 mg/ml and 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice. Figure 2: Stimulation of HEK-Blue™ TNF-a/IL-1b cells by recombinant human HEK-Blue™ TNF- /IL-1 cells are also available in a kit. See below: TNF-a and IL-1b was assessed by measuring the levels of SEAP secreted in the a b supernatant using QUANTI-Blue™.
HEK-Blue™ TNF-a/IL-1b Kit
• HEK-Blue™ TNF-a/IL-1b Cells 5-7 x 106 cells PRODUCT QUANTITY CAT. CODE • Anti-hTNF-a-IgA antibody 100 μg CYTOKINE SIGNALING • Anti-hIL-1b-IgA antibody 100 μg HEK-Blue™ TNFa/IL-1b Cells 5-7 x 106 cells hkb-tnfil1 • QUANTI-Blue™ 1 pouch (100 ml) HEK-Blue™ TNF-a/IL-1b Kit 1 kit hkb-tnfil1-kit • Normocin™ 1 ml (50 mg/ml) • Zeocin™ 100 μl (100 mg/ml)
110 www.invivogen.com/cytokine-sensor-cells www.invivogen.com/cytokine-sensor-cells HEK-Blue™ IL-1b Cells - IL-1b Reporter Cells
➥ Monitor the IL-1b-induced NF-kB and AP-1 pathways ➥ Detection of human and murine IL-1b(a)
Background
Interleukin-1b (IL-1b) is an important mediator of the inflammatory response to infection and injury. IL-1b binds to the type 1 IL-1 receptor (IL-1R) which requires the IL-1 receptor accessory protein (IL-1RAcP) to transduce a signal. IL-1b signaling is mediated by MyD88, IRAK-1/2 and TRAF-6 leading to the activation of NF-kB and AP-1 (figure 1).
Description
HEK-Blue™ IL-1b cells allow to detect bioactive IL-1b by monitoring the activation of the NF-kB and AP-1 pathways. They derive from HEK-Blue™ TNF-a/IL-1b cells in which the TNF-a response has been blocked. Therefore, HEK-Blue™ IL-1b cells respond specifically to IL-1b. They express a SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB and five AP-1 binding sites. Binding of IL-1b to its receptor on the surface of HEK-Blue™ IL-1b cells triggers the IL1-R signaling pathway leading to the activation of NF-kB/ AP-1 and the subsequent production of SEAP. The levels of SEAP in the supernatant can be easily monitored using QUANTI-Blue™. HEK-Blue™ IL-1b cells respond to IL-1b and also IL-1a (data not shown) Figure 1: IL-1b-induced NF-kB signaling pathway. but do not respond to TNF-a (figure 2). Stimulation of HEK-Blue™ IL-1b cells with recombinant human IL-1b can be blocked by the neutralizing monoclonal anti-hIL-1b-IgA antibody (see page 104),
• Detection range for human IL-1b: 100 pg - 100 ng/ml • Detection range for murine IL-1b: 10 ng - 1 μg/ml
HEK-Blue™ IL-1b cells are resistant to Zeocin™ and hygromycin B.
Contents and Storage HEK-Blue™ IL-1b cells are grown in DMEM medium, 2 mM L-glutamine, 10% 4 FBS supplemented with 100 μg/ml Zeocin™ and 200 μg/ml HygroGold™ 6 (ultrapure Hygromycin). Each vial contains 5-7 x 10 cells and is supplied with 10 mg Zeocin™, 10 mg HygroGold™, 50 mg Normocin™ and 1 pouch of QUANTI-Blue™ Cells are shipped on dry ice.
HEK-Blue™ IL-1b cells are also available in a kit. See below:
Figure 2: Stimulation of HEK-Blue™ IL-1b cells by human and murine IL-1b and human TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue™.
HEK-Blue™ IL-1b Kit
• HEK-Blue™ IL-1b Cells 5-7 x 106 cells PRODUCT QUANTITY CAT. CODE • Anti-hIL-1b-IgA antibody 100 μg • QUANTI-Blue™ 1 pouch (100 ml) HEK-Blue™ IL-1b Cells 5-7 x 106 cells hkb-il1b CYTOKINE SIGNALING • Normocin™ 1 ml (50 mg/ml) HEK-Blue™ IL-1b Kit 1 kit hkb-il1b-kit • HygroGold™ 100 μl (100 mg/ml) • Zeocin™ 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells 111 HEK-Blue™ IL-18/IL-1b Cells - IL-18/IL-1b Reporter Cells
➥ Monitor the IL-18/IL-1b-induced NF-kB and AP-1 pathways ➥ Detection of IL-18 and IL-1b(a)
Background
Interleukin-1b (IL-1b) and IL-18 are related pro-inflammatory cytokines that cause a wide variety of biological efffects associated with infection, inflammation and autoimmune processes. IL-1b binds to the type 1 IL-1 receptor which requires the IL-1 receptor accessory protein (IL-1RAcP) to transduce a signal. IL-18 binds to an heterodimeric receptor consisting of IL-18R and IL-18 receptor accessory protein (IL18RAP). IL-1b and IL- 18 share common signaling pathways that involve MyD88, and TRAF-6 leading to the activation of NF-kB and AP-1 (figure 1).
Description
HEK-Blue™ IL-18/IL-1b cells are designed to detect bioactive IL-18 and IL-1b (and IL-1a) by monitoring the activation of the NF-kB and AP-1 pathways. They were generated by stable transfection of HEK-Blue™ IL-1b cells with the gene encoding IL-18RAP. They express a SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB and five AP-1 binding sites. Stimulation of HEK-Blue™ IL-18/IL-1b cells with human IL-18 or IL-1b, but not with human TNF-a, activates the NF-kB and AP-1 pathways Figure 1: IL-18- and IL-1b--induced signaling pathways. triggering the production of SEAP (figure 2). Levels of SEAP in the supernatant can be easily determined with QUANTI-Blue™. Stimulation of HEK-Blue™ IL-18/IL-1b cells with recombinant human IL-18 or IL-1b can be blocked by the neutralizing antibodies anti-hIL-18-IgA and anti-hIL-1b-IgA, respectively (see page 104).
• Detection range for human IL-18: 0.5 - 100 ng/ml • Detection range for human IL-1b: 0.5 - 100 ng/ml
HEK-Blue™ IL-18/IL-1b cells are resistant to blasticidin, Zeocin™ and hygromycin B.
4 Contents and Storage HEK-Blue™ IL-18/IL-1b cells are grown in DMEM medium, 2mM L-glutamine, 10% FBS supplemented with 30 μg/ml blasticidin, 100 μg/ml Zeocin™ and 200 μg/ml HygroGold™ (ultrapure Hygromycin). Each vial 6 contains 5-7 x 10 cells and is supplied with 1 mg blasticidin, 10 mg Zeocin™, 10 mg HygroGold™, 50 mg Normocin™ and 1 pouch of QUANTI-Blue™ Cells are shipped on dry ice. Figure 2: Stimulation of HEK-Blue™ IL-18/IL-1 cells by human IL-18, IL-1 and HEK-Blue™ IL-18/IL-1b cells are also available in a kit, see below: b b TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue™.
HEK-Blue™ IL-18/IL-1b Kit
• HEK-Blue™ IL-18/IL-1b Cells 5-7 x 106 cells • Anti-hIL-18-IgA antibody 100 μg PRODUCT QUANTITY CAT. CODE • Anti-hIL-1b-IgA antibody 100 μg CYTOKINE SIGNALING • QUANTI-Blue™ 1 pouch (100 ml) HEK-Blue™ IL-18/IL-1b Cells 5-7 x 106 cells hkb-il18 ™ • Normocin 1 ml (50 mg/ml) HEK-Blue™ IL-18/IL-1b Kit 1 kit hkb-il18-kit • Blasticidin 100 μl (10 mg/ml) • HygroGold™ 100 μl (100 mg/ml) • Zeocin™ 100 μl (100 mg/ml)
112 www.invivogen.com/cytokine-sensor-cells www.invivogen.com/cytokine-sensor-cells HEK-Blue™ IL-33/IL-1b Cells - IL-33/IL-1b Reporter Cells NEW
➥ Monitor the IL-33/IL-1b-induced NF-kB and AP-1 pathways ➥ Detection of IL-33 and IL-1b(a)
Background
Interleukin-33 (IL-33) is a member of the IL-1 family, a group of cytokines that play important roles in host defense, immune regulation and inflammation. IL-33 mediates its biological effects through ST2 (also known as IL1RL1), a receptor expressed on Th2 and mast cells. IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. IL-33 signaling leads to the activation of NF-kB and MAP kinases, and the production of Th2- associated cytokines.
Description
HEK-Blue™ IL-33/IL-1b cells are designed to detect bioactive IL-33 and IL-1b (and IL-1a) by monitoring the activation of the NF-kB and AP-1 pathways. They were generated by stable transfection of HEK-Blue™ IL-1b cells with the IL1RL1 gene. They express a SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB and five AP-1 binding sites. Stimulation of HEK-Blue™ IL-33/IL-1b cells with human IL-33 or IL-1b, but not with IL-18 nor TNF-a, activates the NF-kB and AP-1 pathways Figure 1: IL-33- and IL-1b--induced signaling pathways. triggering the production of SEAP (figure 2). Levels of SEAP in the supernatant can be easily determined with QUANTI-Blue™. Stimulation of HEK-Blue™ IL-33/IL-1b cells with recombinant human IL-1b can be blocked by using the neutralizing antibody anti-hIL-1b-IgA.
• Detection range for human IL-33: 0.5 - 100 ng/ml • Detection range for human IL-1b: 0.5 - 100 ng/ml
HEK-Blue™ IL-33/IL-1b cells are resistant to blasticidin, Zeocin™ and hygromycin B.
Contents and Storage
™ 4 HEK-Blue IL-33/IL-1b cells are grown in DMEM medium, 2mM L-glutamine, 10% FBS supplemented with 30 μg/ml blasticidin, 100 μg/ml Zeocin™ and 200 μg/ml HygroGold™ (ultrapure Hygromycin). Each vial 6 contains 5-7 x 10 cells and is supplied with 1 mg blasticidin, 10 mg Zeocin™, 10 mg HygroGold™, 50 mg Normocin™ and 1 pouch of QUANTI-Blue™ Cells are shipped on dry ice.
Figure 2: Stimulation of HEK-Blue™ IL-33/IL-1b cells by human IL-33, IL-1b, IL-18 and TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue™.
Related Products
Anti-hIL-1b-IgA , page 104 Blasticidin, page 17 Normocin™, page 11 HygroGold™, page 18 QUANTI-Blue™, page 14 Zeocin™, page 18 PRODUCT QUANTITY CAT. CODE
HEK-Blue™ IL-33/IL-1b Cells 5-7 x 106 cells hkb-il33 CYTOKINE SIGNALING
www.invivogen.com/cytokine-sensor-cells 113 HEK-Blue™ TGF-b Cells - TGF-b Reporter Cells
➥ Monitor the TGF-b/Smad pathway ➥ Detection of human TGF-b
Background
Tumor growth factor-beta (TGF-b) belongs to a family of structurally related cytokines that regulate a plethora of cellular functions, such as proliferation, apoptosis, differentiation and migration. TGF-b binds to a type II receptor which recruits and activates a type I receptor. The type I receptor then phosphorylates receptor-regulated Smads (R-Smads), such as Smad2 and Smad3, which associate with Smad4. R-Smad/Smad4 complexes accumulate in the nucleus where they regulate the transcription of target genes.
Description
HEK-Blue™ TGF-b cells allow the detection of bioactive TGF-b by monitoring the activation of the TGF-b/Smad pathway. They were generated by stable transfection of HEK293 cells with the human TGFBRI, Smad3 and Smad4 genes. They further express a SEAP reporter gene under the control of the b-globin minimal promoter fused to three Smad3/4-binding elements (SBE). Stimulation of HEK-Blue™ TGF-b cells with TGF-b induces the activation of the TGF-b/Smad signaling pathway leading to the formation of a Figure 1: TGF-b-induced Smad signaling pathway Smad3/Smad4 complex. The heterocomplex enters the nucleus and binds SBE sites inducing the production of SEAP (figure 1). The quantity of SEAP secreted in the supernatant can be easily assessed using QUANTI-Blue™ (figure 2). TGF-b-mediated SEAP production can be blocked using a neutralizing antibody, such as anti-hTGF-b-IgA (see page 104).
• Detection range for TGF-b: 0.1 - 10 ng/ml
HEK-Blue™ TGF-b cells are resistant to blasticidin, hygromycin and Zeocin™.
Contents and Storage
4 ™ HEK-Blue TGF-b cells are grown in DMEM medium, 2 mM L-glutamine, 10% FBS supplemented with 30 μg/ml blasticidin, 200 µg/ml HygroGold™ (ultrapure hygromycin) and 100 µg/ml Zeocin™. Cells are provided frozen in a cryotube containing 5-7 x 106 cells and supplied with 100 µl of blasticidin at 10 mg/ml, 100 µl of HygroGold™ at 100 mg/ml, 100 µl of Zeocin™ at 100 mg/ml and 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice. HEK-Blue™ TGF-b cells are also available in a kit, see below: Figure 2: Stimulation of HEK-Blue™ TGF-b cells by human TGF-b was assessed by measuring the levels of SEAP using QUANTI-Blue™.
HEK-Blue™ CD40L Kit
• HEK-Blue™ TGF-b Cells 5-7 x 106 cells • Anti-hTGF-b-IgA antibody 100 μg PRODUCT QUANTITY CAT. CODE • QUANTI-Blue™ 1 pouch (100 ml)
CYTOKINE SIGNALING • Normocin™ 1 ml (50 mg/ml) HEK-Blue™ TGF-b Cells 5-7 x 106 cells hkb-tgfb • Blasticidin 100 μl (10 mg/ml) HEK-Blue™ TGF-b Kit 1 kit hkb-tgfb-kit • HygroGold™ 100 μl (100 mg/ml) • Zeocin™ 100 μl (100 mg/ml)
114 www.invivogen.com/cytokine-sensor-cells www.invivogen.com/cytokine-sensor-cells HEK-Blue™ CD40L Cells - CD40L Reporter Cells
➥ Study CD40L-CD40 interactions ➥ Screen for molecules that interfere with CD40L-CD40 cross-talk
Background
CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) family of cell surface interaction molecules. It is mainly expressed in CD4+-T cells and interacts with CD40 on antigen-presenting cells to regulate both humoral and cellular immune responses. CD40L-CD40 interactions are thought to play an important role in the pathogenesis of certain diseases, including AIDS, Alzheimer’s disease and rheumatoid arthritis.
Description
HEK-Blue™ CD40L cells can serve to measure the bioactivity of CD40L through the secretion of embryonic alkaline phosphatase (SEAP) upon NF-kB activation. These cells were generated by stable transfection of HEK293 cells with the human CD40 gene and an NF-kB-inducible SEAP construct. The SEAP construct consists of the SEAP reporter gene under the control of the IFN-b minimal promoter fused to five NF-kB binding sites. Binding of CD40L to its receptor CD40 triggers a signaling cascade leading to the activation of NF-kB and the subsequent production of SEAP (figure 1). CD40L-CD40 interaction can be monitored by assessing Figure 1: CD40L-induced NF-kB signaling pathway. the levels of SEAP using a SEAP detection assay, such as QUANTI-Blue™ (figure 2). HEK293 cells express endogenously the receptors for the cytokines IL-1b and TNF-a which share a common signaling pathway with CD40L. Consequently, HEK-Blue™ CD40L cells also respond to IL-1b and TNF-a. IL-1b- and TNF-a-mediated SEAP production can be blocked using neutralizing antibodies, such as anti-hIL-1b-IgA and anti-hTNF-a-IgA antibodies respectively (see page 104).
• Detection range for CD40L: 5 ng - 1 μg/ml
HEK-Blue™ CD40L cells are resistant to the selective antibiotics blasticidin and Zeocin™.
Contents and Storage 4
HEK-Blue™ CD40L cells are grown in DMEM medium, 2mM L-glutamine, 10% FBS supplemented with 100 μg/ml Zeocin™ and 30 μg/ml blasticidin. 6 Each vial contains 5-7 x 10 cells and is supplied with 10 mg Zeocin™, 1 mg blasticidin, 50 mg Normocin™ and 1 pouch of QUANTI-Blue™. Cells are shipped on dry ice. ™ HEK-Blue CD40L cells are also available in a kit, see below: Figure 2: Stimulation of HEK-Blue™ CD40L cells by human CD40L was assessed by measuring the levels of SEAP using QUANTI-Blue™.
HEK-Blue™ CD40L Kit
• HEK-Blue™ CD40L Cells 5-7 x 106 cells • Anti-hCD40L-IgA antibody 100 μg PRODUCT QUANTITY CAT. CODE • QUANTI-Blue™ 1 pouch (100 ml)
• Normocin™ 1 ml (50 mg/ml) HEK-Blue™ CD40L Cells 5-7 x 106 cells hkb-cd40 CYTOKINE SIGNALING • Blasticidin 100 μl (10 mg/ml) HEK-Blue™ CD40L Kit 1 kit hkb-cd40-kit • Zeocin™ 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells 115 HEK-Blue™ Cytokine Kits
PRODUCT CONTENTS CAT. CODE
HEK-Blue™ IL-4/IL-13 Kit • HEK-Blue™ IL-4/IL-13 Cells (5-7 x 106 cells) • Normocin™ (1 ml @ 50 mg/ml) hkb-stat6-kit • Anti-hIL-4-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml) • Anti-hIL-13-IgA neutralizing antibody (100 μg) • Zeocin™ (100 μl @ 100 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml)
HEK-Blue™ IL-6 Kit • HEK-Blue™ IL-6 Cells (5-7 x 106 cells) • Normocin™ (1 ml @ 50 mg/ml) hkb-il6-kit • Anti-hIL-6-IgA neutralizing antibody (100 μg) • HygroGold™ (100 μl @ 100 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml) • Zeocin™ (100 μl @ 100 mg/ml)
HEK-Blue™ TNF-a/IL-1b Kit • HEK-Blue™ TNF-a/IL-1b Cells (5-7 x 106 cells) • QUANTI-Blue™ (1 pouch, 100 ml) hkb-tnfil1-kit • Anti-hTNF-a-IgA neutralizing antibody (100 μg) • Normocin™ (1 ml @ 50 mg/ml) • Anti-hIL-1b-IgA neutralizing antibody (100 μg) • Zeocin™ (100 μl @ 100 mg/ml)
HEK-Blue™ IL-1b Kit • HEK-Blue™ IL-1b Cells (5-7 x 106 cells) • Normocin™ (1 ml @ 50 mg/ml) hkb-il1b-kit • Anti-hIL-1b-IgA neutralizing antibody (100 μg) • HygroGold™ (100 μl @ 100 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml) • Zeocin™ (100 μl @ 100 mg/ml)
HEK-Blue™ IL-18/IL-1b Kit • HEK-Blue™ IL-18/IL-1b Cells (5-7 x 106 cells) • Normocin™ (1 ml @ 50 mg/ml) hkb-il18-kit • Anti-hIL-18-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml) • Anti-hIL-1b-IgA neutralizing antibody (100 μg) • HygroGold™ (100 μl @ 100 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml) • Zeocin™ (100 μl @ 100 mg/ml)
HEK-Blue™ TGF-b Kit • HEK-Blue™ TGF-b Cells (5-7 x 106 cells) • Blasticidin (100 μl @ 10 mg/ml) hkb-tgfb-kit • Anti-hTGF-b-IgA neutralizing antibody (100 μg) • HygroGold™ (100 μl @ 100 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml) • Zeocin™ (100 μl @ 100 mg/ml) • Normocin™ (1 ml @ 50 mg/ml)
HEK-Blue™ CD40L Kit • HEK-Blue™ CD40L Cells (5-7 x 106 cells) • Normocin™ (1 ml @ 50 mg/ml) hkb-cd40-kit • Anti-hCD40L-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml) • QUANTI-Blue™ (1 pouch, 100 ml) • Zeocin™ (100 μl @ 100 mg/ml)
Contents and Storage
HEK-Blue™ Cytokine cells are shipped on dry ice. Thaw immediately or store in liquid nitrogen. Other products are shipped at room temperature and should 4 be stored at -20°C. CYTOKINE SIGNALING
116 www.invivogen.com/cytokine-sensor-cells