IL-6 Reporter HEK 293 Cells

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Interleukin-6 Reporter Cells

Signaling pathway in HEK-Blue™ IL-6 cells

HEK-Blue™ IL-6 cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive human interleukin-6 (IL-6) by monitoring the activation of the JAK/STAT3 pathway. They can also be used for screening antibodies or small molecule inhibitors targeting the IL-6 pathway.

IL-6 is a multifunctional cytokine that plays a key role in the regulation of the immune response, hematopoiesis, and inflammation. It is one of the most important mediators of fever and of the acute phase response [1, 2].

More details

Cell line description

HEK-Blue™ IL-6 cells were generated by stable transfection with the genes encoding the IL-6Rα chain of the human IL-6 receptor, as well as human STAT3 to obtain a fully active IL-6 signaling pathway. The gp130 chain of the IL-6 receptor is endogenously expressed. They were further transfected with a STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-6 to its receptor triggers a signaling cascade leading to the activation of STAT3 and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ IL-6 cells respond to recombinant human IL-6 (see figures).  Of note, these cells also exhibit a moderate response to human IFN-γ. They can also be used for screening and release assays of molecules that inhibit IL-6 signaling, such as tocilizumab, a monoclonal antibody targeting the IL-6 receptor (IL-6R).

Key features

• Fully functional IL-6 signaling pathway
• Readily assessable STAT3-inducible SEAP reporter activity
• Strong response to human IL-6

Applications

• Detection and quantification of hIL-6 and mIL-6 activity
• Screening of anti-IL-6 and anti-IL-6 receptor antibodies (e.g. tocilizumab)
• Screening of small molecule inhibitors of the IL-6 pathway

References:

1. Johnson DE. et al., 2018. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 15(4):234.
2. Hunter CA. & Jones SA., 2015. IL-6 as a keystone cytokine in health and disease. Nat Immunol. 16(5):448.

Figures

Dose-response of HEK-Blue™ IL-6 cells to recombinant human IL‑6. Cells were stimulated with increasing concentration of recombinant human IL-6. After overnight incubation, the STAT3 response was determined using QUANTI‑Blue™, a SEAP detection reagent, and reading the optical density (OD)at 630 nm.

Dose-dependent inhibition of HEK-Blue™ IL-6 cells response using tocilizumab biosimilar. Anti-hIL6R-To-hIgG1 (0 - 1 μg/ml) was incubated with HEK-Blue™ IL-6 cells for 3 hours. Following this, recombinant human IL-6 (0.3 ng/ml) was added to the cells. After overnight incubation, STAT3‑dependent SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as percentage (%) of the maximum response.

Cytokine response profile of HEK-Blue™ IL-6 cells. Cells were stimulated with various human recombinant cytokines; 3 ng/ml IL-1β, IL-4, IL‑6, IL-13, IL-18, IFN-γ, TGF-β, TNF-α or 1x10⁴ IU/ml IFN-α and IFN-β. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™.

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 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 IL-6

Detection range:

• Detection range for human IL-6: 0.03 ng/ml - 10 ng/ml

Quality Control:

• SEAP reporter activity in response to IL-6 is validated using functional assays.
• The stability for 20 passages following thawing is confirmed.
• These cells are tested for mycoplasma contamination. 

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 2 x 1 ml of HEK-Blue™ Selection (250x concentrate)
• 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

The proinflammatory cytokine IL-6 is one of the most important mediators of fever and of the acute phase response [1, 2].

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 dimerization and translocation to the nucleus of STAT3 where it binds enhancer elements of IL-6-inducible genes [3].

In HEK-Blue™ IL-6 cells activation of the JAK/STAT3 pathway with IL-6 leads to the production of SEAP.

1. Kang S. et al., 2015. Therapeutic uses of anti-interleukin-6 receptor antibody. Int. Immunol. 27: 21-9.
2. Akira S. et al., 1990. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J. 4(11):2860-7.
3. Murray P., 2007. The JAK-STAT Signaling Pathway: Input and Output Integration. J. Immunol. 178:2623-9.

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