IL-12 Reporter HEK 293 Cells

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IL-12 Reporter Cells

Signaling pathway in HEK-Blue™ IL-12 cells

HEK-Blue™ IL-12 cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive human and murine interleukin-12 (IL-12) by monitoring the activation of the JAK/STAT4 pathway. They can also be used for screening antibodies or small molecule inhibitors targeting the IL-12 pathway.

IL-12 is a pro-inflammatory cytokine that regulates T-cell and natural killer-cell responses, as well as inducing the production of interferon-γ (IFN-γ) [1-3].

 More details

Cell line description

HEK-Blue™ IL-12 cells were generated by stable transfection with the genes encoding for the human IL-12 receptor (IL-12Rβ1 and IL-12Rβ2 chains), JAK2, and a STAT4-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-12 to its receptor triggers a signaling cascade leading to the activation of STAT4 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-12 cells detect human and murine IL-12 (see figures).

Key features

• Fully functional IL-12 signaling pathway
• Readily assessable STAT4-inducible SEAP reporter activity
• Strong response to human (h) and mouse (m) IL-12

Applications

• Detection and quantification of hIL-12 and mIL-12 activity
• Screening of anti-IL-12 and anti-IL-12 receptor antibodies
• Screening of small molecule inhibitors of the IL-12 pathway

References:

1. Teng M.W. et al., 2015. IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases. Nat Med. 21(7):719-29.
2. Vignali DA. & Kuchroo VK., 2012. IL-12 family cytokines: immunological playmakers. Nat Immunol. 13(8):722-8.
3. Trinchieri G., 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 3(2):133-46.

Figures

Response of HEK-Blue™ IL-12 cells to increasing concentrations of recombinant human IL-12 and murine IL-12.

After a 24h incubation, the levels of SEAP were determined using QUANTI-Blue™ Solution,  a SEAP detection reagent, and by reading the optical density (OD) at 630 nm.
 

Ligand	EC50	Response Ratio
Human IL-12	4.2 ng/ml	13
Murine IL-12	4.1 ng/ml	11

 

The response ratio was calculated by dividing the OD at 630 nm for the treated cells by the OD at 630 nm for the untreated cells.

 

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, 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 IL-12 and mouse IL-12

Detection range:

• Detection range for human IL-12: 1 ng/ml - 100 ng/ml
• Detection range for murine IL-12: 1 ng/ml - 100 ng/ml

Quality Control:

• SEAP reporter activity in response to IL-12 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 HEK-Blue™ Selection (250x concentrate)
• 1 ml 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

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor (CLMF) and natural killer cell stimulatory factor (NKSF), is primarily produced by dendritic cells and macrophages.

It promotes the development of Th1 responses and is a powerful inducer of interferon-gamma (IFN-γ) [1-3].

IL-12 exerts its biological effect following binding to the IL-12 receptor (IL-12R), which is a heterodimeric receptor composed of two subunits, IL-12R-β1 and IL-12R-β2.

The binding of IL-12 to its receptor triggers a signaling pathway involving TyK2 (tyrosine kinase 2), JAK2 (Janus kinase 2), and STAT4 (signal transducer and activator of transcription 4) which results in the production of IFN-γ.

In HEK-Blue™ IL-12 cells, activation of the STAT4 pathway with IL-12 leads to the production of SEAP.

1. Teng M.W. et al., 2015. IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases. Nat Med. 21(7):719-29.
2. Vignali DA. & Kuchroo VK., 2012. IL-12 family cytokines: immunological playmakers. Nat Immunol. 13(8):722-8.
3. Trinchieri G., 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 3(2):133-46.

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