IL-6 Reporter HEK 293 Cells

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IL-6 Reporter Cells

HEK-Blue™ IL-6 Cells signaling pathway

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. 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 detect human IL-6 (see figures). Of note, these cells also exhibit a moderate response to human IFN-γ (see figures). Stimulation of HEK-Blue™ IL-6 cells with recombinant human IL-6 can be blocked by anti-hIL-6-IgG (see figures).

Key features:

• Fully functional IL-6 signaling pathway
• Readily assessable STAT3-inducible SEAP reporter activity

Applications:

• Detection of human IL-6
• Screening of anti-IL-6 or anti-IL-6R antibodies

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 NF-κB response was determined using QUANTI‑Blue™, a SEAP detection reagent, and reading the optical density (OD)at 630 nm.

Dose-dependent inhibition of IL-6 response using Anti-hIL-6-IgG neutralizing antibody in HEK-Blue™ IL-6 cells. Anti-hIL-6-IgG was incubated with 1 ng/ml of hIL-6 for 30 minutes prior to the addition of HEK‑Blue™ IL-6 cells. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™. Data represent % inhibition of reporter activity.

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, 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™

Guaranteed mycoplasma-free

Specificity: human IL-6

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

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

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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