IL-10 Reporter HEK 293 Cells

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IL-10 Reporter Cells

Signaling pathway in HEK-Blue™ IL-10 cells

HEK-Blue™ IL-10 cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human interleukin 10 (IL-10) by monitoring the activation of the STAT3 pathway. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the IL-10 pathway.

IL-10 is an anti-inflammatory cytokine with a crucial role in preventing inflammatory and autoimmune pathologies [1, 2].

 More details

Cell line description

HEK-Blue™ IL-10 cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with the genes encoding hIL-10R α and β chains, human STAT3, and the STAT3-inducible SEAP (secreted embryonic alkaline phosphatase) reporter.

The binding of IL-10 to its receptor on the surface of HEK-Blue™ IL-10 cells triggers JAK1/STAT3 signaling 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-10 cells detect human (h) IL-10 and, to a lesser extent, murine (m) IL-10 (see figures). These cells are not responsive to hIL-1β, hIL-2, hIL-6, hIL-12, hIL-22, hIL-23, hIFN-α, hIFN-β, hIFN-γ, and hTNF-α (see figures).

Key features

• Fully functional IL-10 signaling pathway
• Readily assessable STAT3-inducible SEAP reporter activity
• Response to human (h) IL-10 and, to a weaker extent, murine (m) IL-10 
• No response to hIL-22

Applications

• Detection and quantification of human and murine IL-10 activitiy
• Screening of anti-IL-10 and anti-IL-10R antibodies
• Screening of small molecule inhibitors of the IL-10 pathway

References:

1. Saraiva M, et al., 2019. Biology and therapeutic potential of interleukin-10. J.  Exp. Med. doi: 10.1084/jem.20190418.
2. Ouyang W & O’Garra A, 2019. IL-10 family cytokines IL-10 and IL-22: from basic science to clinical translation. Immunity. 50:871-91.

Figures

Dose-response of HEK-Blue™ IL‑10 cells to recombinant hIL‑10. Cells were stimulated with increasing concentrations of recombinant human IL-10. After overnight incubation, the STAT3 response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 630 nm. EC50 value is shown as mean ± SD

Response of HEK‑Blue™ IL ‑10 cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines:1 ng/ml of hIL-10, 30 ng/ml of mIL-10, 1 ng/ml of hIL-1β, hIL-2, hIL-6,hIL-12, hIL-22, hIL-23, hIFN-γ, hTNF-α, or 102 U/ml hIFN-α or -β. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™Solution. The optical density (OD) at 630 nm is shown as mean ± SEM.

Dose‑dependent inhibition of HEK‑Blue™ IL ‑10 cellular response using a neutralizing antibody against the hIL‑10 receptor (hIL10R).
The anti-hIL10R antibody was incubated with the cells for 30 minutes prior to the addition of hIL-10 (0.5 ng/ml). After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data represent % of maximal reporter activity without the anti-hIL10R antibody

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B Gold, 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 and mouse IL-10

Detection range:

• 0.3 - 30 ng/ml for human IL-10
• 25 - 1000 ng/ml for murine IL-10

Contents

• 3-7 x 10⁶ HEK-Blue™ IL-10 cells in a cryovial or shipping flask
• 2 x 1 ml of HEK-Blue™ Selection (250x concentrate)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasma, bacteria, and fungi.
• 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 10 (IL-10), is the founding member of the IL-10 cytokine family, which comprises IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A (IFN-λ2), IL-28B (IFN-λ3), and IL-29 (IFN-λ1)1. IL-10 was originally depicted as a key mediator of anti-inflammatory responses. While the immunosuppressive action of IL-10 is illustrated by its genetic association to inflammatory bowel disease, or its upregulated expression in chronic infections, an unanticipated IL-10 protective role has been reported in cancer [1].
IL-10 also plays important homeostatic roles in non-immune cells, including neurons, adipocytes, and epithelial cells [1]. The manipulation of IL-10 production and signaling has thus become an attractive therapeutic strategy [2].
Upon IL-10 binding, the IL-10Rα chain oligomerizes with the IL-10Rβ chain. This in turn allows the phosphorylation of JAK1 (Janus kinase 1) and TyK2 (Tyrosine kinase 2), and the subsequent activation of STAT3 (Signal transducer and activator of transcription 3) [1]. Importantly, STAT3 is also activated downstream of other cytokine receptors. How the cells trigger distinct and specific gene expression in response to cytokines signaling through the same transcription factor is not fully understood yet [1].

1. Saraiva M, et al., 2019. Biology and therapeutic potential of interleukin-10. J.  Exp. Med. doi: 10.1084/jem.20190418.
2. Ouyang W & O’Garra A, 2019. IL-10 family cytokines IL-10 and IL-22: from basic science to clinical translation. Immunity. 50:871-91.

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