TNF-α Reporter HEK 293 Cells

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TNF-α Reporter Cells

Signaling pathway in HEK-Blue™ TNF-α cells

HEK-Blue™ TNF-α cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human and murine tumor necrosis factor-alpha (TNF-α) by monitoring the activation of the NF-κB and AP-1 pathways. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the TNF-α pathway. 

TNF-α is a multi-functional pro-inflammatory cytokine involved in regulating a wide spectrum of biological processes, such as cell proliferation, differentiation, and apoptosis [1]. 

 More details

Cell line description

HEK-Blue™ TNF-α cells were generated by stable transfection with the genes encoding for the human TNF-α receptor (TNFR1 and TNFR2 chains), as well as an NF-κB/AP-1-inducible SEAP secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of TNF-α to its receptor triggers a signaling cascade leading to NF-κB/AP1 activation and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. 

HEK-Blue™ TNF-α cells respond to recombinant human TNF-α, as well as recombinant mouse TNF-α (see figures). Of note, these cells are not responsive to human IL-1β. They can also be used for screening and release assay of molecules that inhibit TNF-α signaling, such as adalimumab, a monoclonal antibody targeting TNF-α.

Key features

• Fully functional TNF-α signaling pathway
• Readily assessable NF-κB/AP-1-inducible SEAP reporter activity
• Strong response to human (h) and murine (m) TNF-α
• No response to hIL-1β

Applications

• Detection and quantification of human and mouse TNF-α activity
• Screening of anti-TNF-α or anti-TNFR antibodies (e.g. adalimumab)
• Screening and release assay of small molecule inhibitors of the TNF-α pathway

Reference:

1. Steeland S, Libert C, Vandenbroucke RE. 2018. A New Venue of TNF Targeting. Int J Mol Sci.;19(5):1442. 

Figures

Dose-response of HEK-Blue™ TNF-α cells to recombinant human (h) or murine (m) TNF-α cytokine. Cells were stimulated with increasing concentrations of recombinant hTNF-α or mTNF-α. After overnight incubation, the NF-κB-induced SEAP activity was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Response profile of HEK-Blue™ TNF-α cells. Cells were incubated for 24 hours with cytokines and various TLR agonists: hTNF-α (100 pg/ml), mTNF-α (100 pg/ml), and hIL-1β (10 ng/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-dependent inhibition of HEK-Blue™ TNF-α cells response using adalimumab biosimilar. A serial dilution of Anti-hTNF-α-IgG1 monoclonal antibody (mAb) was incubated with 30 pg/ml of recombinant human TNF-α for 30 minutes prior to the addition of the HEK-Blue™ TNF-α cells. After overnight incubation, the NF-κB response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as percentage of activity (mean ± SEM).

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®

Specificity: Detects human and mouse TNF-α

Detection range:

• 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)

Quality Control:

• SEAP reporter activity in response to TNF-α is validated using functional assays.
• The stability for 20 passages following thawing is confirmed.
• These cells are tested for mycoplasma contamination. 

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

Contents

• 1 vial containing 3-7 x 10⁶ 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 cytokine involved in necrotic and apoptotic cell death, cellular differentiation, inflammation, and regulation of immune cell activity [1]. Notably, deregulated TNF-α production has been implicated in a variety of conditions, including autoimmune and inflammatory diseases [1].

TNF-α is mainly produced by activated monocytes, macrophages, and T cells. It is first synthesized as a membrane-bound molecule that forms a compact homotrimer through non-covalent interactions. The trimeric membrane-bound form is cleaved by tumor necrosis factor-alpha converting enzyme (TACE) releasing the soluble trimer [2]. Both the membrane-bound and soluble TNF-α bind homotrimeric transmembrane receptors, TNFR1 or TNFR2, triggering signaling pathways that involve TRADD, TRAF2, and RIP, and leading to the activation of NF-κB and MAPK pathways.

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. Steeland S. et al., 2018. A new venue of TNF targeting. Int. J. Mol. Sci. 19:1442.
2. Brenner D. et al., 2015. Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol. 15(6):362-74.

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