IL-10 Reporter HEK 293 Cells

IL-10 responsive STAT3-SEAP reporter assay

Signaling pathway in HEK-Blue™ IL-10 cells

HEK-Blue™ IL-10 cells are designed to monitor human IL-10-induced STAT3 stimulation or inhibition through SEAP detection. This colorimetric bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies.

HEK-Blue™ IL-10 cells respond strongly to recombinant human IL-10, and to a lower extent to mouse (m)IL-10 (see figures). The reliable and consistent performance of HEK-Blue™ IL-10 cells makes them suitable for release assays of therapeutic molecules that inhibit IL-10 signaling, such as monoclonal antibodies.

Key features

• Readily assessable STAT3-inducible SEAP reporter activity
• Convenient readout using  QUANTI-Blue™ Solution
• Response to human (h) IL-10 and, to a weaker extent, mouse (m) IL-10 
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

Interleukin 10 (IL-10) is an anti-inflammatory cytokine with a crucial role in preventing inflammatory and autoimmune pathologies. While it is known for its immunosuppressive role in conditions like inflammatory bowel disease and chronic infections, it also shows a surprising protective effect in cancer.

 More details

Note: A new clone is provided with an improved IL-10 response. The cat code has been changed accordingly (hkb-il10v2).

Figures

Dose-response of HEK-Blue™ IL-10 cells to recombinant IL-10. Cells were stimulated with increasing concentrations of recombinant human IL-10 (hIL-10) and mouse IL-10 (mIL-10). After overnight incubation, the STAT3-induced SEAP activity was determined using QUANTI-Blue™, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Response of HEK-Blue™ IL-10 cells to a panel of cytokines. Cells were stimulated with various human and mouse recombinant cytokines: 10 ng/ml of hIL-10 and mIL-10, 100 ng/ml of hIL-1β, hIL-6, hIL-19, hIL-20, hIL-22, hIL-24, hIL-26, hIL-27, hTNF-α, and 100 U/ml hIFN-γ. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: IL-10

Specificity: Human, mouse

Reporter gene: SEAP

Antibiotic resistance: Blasticidin, Hygromycin, Zeocin®

Detection ranges: 

• Human IL-10: 0.3 - 30 ng/ml
• Mouse IL-10: 25 - 1000 ng/ml

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

Contents

HEK-Blue™ IL-10 Cells (hkb-il10v2)

• 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)

HEK-Blue™ IL-10 vial (hkb-il10v2-av)

• 1 vial containing 3-7 x 10⁶ cells

 Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Notification:  Reference #hkb-il10-av can only be ordered together with reference #hkb-il10v2.

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).

IL-10 background

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 with 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 cytokine 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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