TNF-α Dual Reporter HEK 293 Cells

HEK-Dual™ TNF-α cells

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

HEK-Dual™ TNF-α cells enable the detection of bioactive human tumor necrosis factor-alpha (TNF-α) by monitoring the activation of the NF-κB pathway. These cells express two different NF-κB-inducible reporters, secreted embryonic alkaline phosphatase (SEAP) or the secreted Lucia luciferase.

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-Dual™ TNF-α cells were derived from the human embryonic kidney 293 cell line by stable co-transfection of two NF-κB-inducible reporter constructs, the SEAP or secreted Lucia luciferase gene. Due to the stable knockout of the MyD88 gene, HEK-Dual™ TNF-α cells do not respond to IL-1β. As a result, HEK-Dual™ TNF-α cells allow the specific study of TNF-α-induced NF-κB activation by monitoring the activity of either SEAP or secreted Lucia luciferase, each reporter system presenting specific advantages. Both reporter genes are under the control of the same NF-κB-inducible promoter consisting of an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site.

Stimulation of HEK-Dual™ TNF-α cells with TNF-α triggers the activation of the NF-κB-inducible promoter and the production of SEAP as well as Lucia luciferase. These reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™, a luciferase detection reagent.

Features of HEK-Dual™ TNF-α cells:

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

Applications of HEK-Dual™ TNF-α cells:

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

Specifications

Antibiotic resistance: Zeocin®

Growth medium: DMEM medium, 2mM L-glutamine,10% FBS supplemented with 100 µg/ml Normocin and 100 µg/ml Zeocin®

Guaranteed mycoplasma-free

• Detection range for human TNF-α using QUANTI-Luc™: 0.5 ng/ml - 1 µg/ml
• Detection range for human TNF-α using QUANTI-Blue™: 1 ng/ml - 1 µg/ml

These products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial of HEK-Dual™ TNF-α Cells (3-7 x 10e6 cells)
• 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)
• 1 pouch of QUANTI-Luc™ (Lucia luciferase detection medium)

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-Dual™ 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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