TNF-α Reporter HEK 293 Cells

TNF-α signaling pathway in HEK-Blue™ TNF-α cells

Human and murine TNF-α Reporter Cells

HEK-Blue™ TNF-α cells enable the detection of bioactive human and murine tumor necrosis factor-alpha (TNF-α) by monitoring the activation of the NF-κB pathway.
TNF-α is a multi-functional pro-inflammatory cytokine involved in the regulation of a wide spectrum of biological processes, such as cell proliferation, differentiation, and apoptosis [1]. Notably, deregulated TNF-α production has been implicated in a variety of conditions, including autoimmune and inflammatory diseases [1].

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Cell line description:

HEK-Blue™ TNF-α cells were generated by stable transfection of human embryonic kidney HEK293 cell line with a SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB (and five AP-1) binding sites. They were further rendered unresponsive to IL-1β by knocking-out the MyD88 gene. Stimulation of HEK-Blue™ TNF-α cells with TNF-α triggers the activation of the NF-κB-inducible promoter and the production of SEAP.

Levels of SEAP in the supernatant can be easily determined using QUANTI-Blue™ Soultion, a reagent that turns purple/blue in the presence of SEAP, by reading the OD at 620-655 nm.

HEK-Blue™ TNF-α cells are resistant to Puromycin and Zeocin® .

Features of HEK-Blue™ TNF-α cells:

• Fully functional TNF-α signaling pathway
• Do not respond to IL-1β
• Readily assessable SEAP reporter activity
• Functionally tested and guaranteed mycoplasma-free

Applications of HEK-Blue™ TNF-α cells:

• Detection of human TNF-α
• Screening of anti-TNF-α antibodies

Reference:

1. Sedger L. & McDermott M., 2014. TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 25(4):453-72.

Figures

Response of HEK-Blue™ TNF-α cells to TNF-α and IL-1β. Cells were incubated with increasing concentrations of human and murine TNF-α and human IL-1β. After a 24h incubation, NF-κB/AP-1 activation was assessed by measuring the levels of SEAP in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. 

Specifications

Antibiotic resistance: Puromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v)  heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Quality Control:

• Reporter activity is validated by stimulating the cells with human and murine TNF-α.
• The cells are guaranteed mycoplasma-free.

Detects human and murine TNF-α

• hTNF-α EC50: 0.01 ng/ml (in medium) or 0.7 ng/ml (in water)
• mTNF-α EC50: 0.1 ng/ml (in medium) or 3 ng/ml (in water)

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial of HEK-Blue™ TNF-α cells (3-7 x 10e6 cells)
• 1 ml of Puromycin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine produced by several types of cells, predominantly activated macrophages [1.] TNF-α plays an important role in the immune response to microbial invasions and in the necrosis of specific tumors. Of note,  as a potent mediator of inflammation, TNF-α has implicated in the pathogenesis of several autoimmune and inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease [1,2].
TNF-α exists in two forms; a type II transmembrane protein and a mature soluble protein. The TNF-α transmembrane protein is proteolytically cleaved to yield a soluble protein [3], which subsequently forms a non-covalently linked homotrimer in solution. TNF-α binds two receptors TNFR1 and TNFR2 inducing signaling that involves TRADD, TRAF2, and RIP, and leads to the activation of the NF-κB and the MAPK pathways [4]. 

Interleukin 1 beta (IL-1β) is another inflammatory cytokine that triggers these pathways following the binding to its receptor IL-1RI and the recruitment of MyD88. Both TNF-α and IL-1β receptors are expressed in HEK293 cells. HEK-Blue™ TNF-α Cells are rendered unresponsive to IL-1β by stable knock-out of the MyD88 gene.

1. Sedger L. & McDermott M., 2014. TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 25(4):453-72.
2. Li P. et al., 2017. Drugs for Autoimmune Inflammatory Diseases: From Small Molecule Compounds to Anti-TNF Biologics.Front Pharmacol .8:460.
3. Kriegler M. et al., 1988. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell. 53(1):45-53.
4. Wajant H. et al., 2003. Tumor necrosis factor signaling. Cell Death Differ. 2003 10(1):45-65.

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