IL-36 Reporter HEK 293 Cells

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Interleukin-36 Reporter Cells

Signaling pathway in HEK-Blue™ IL-36 cells

HEK-Blue™ IL-36 cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive interleukin-36 (IL-36) by monitoring the activation of NF-κB/AP-1 pathways. Three isoforms, IL-36α, IL-36β, and IL-36γ, mediate pro-inflammatory functions, while a fourth one, IL-36Ra, acts as an antagonist [1,2].

 More details

Cell line description:

HEK-Blue™ IL-36 cells were generated by stable transfection with the genes encoding for the human IL-36 receptor (IL-1R6 and IL-1RAcP chains), and an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-36 to its receptor triggers a signaling cascade leading to the NF-κB/AP-1 activation 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-36 cells respond to human (h) but not murine (m) IL-36 agonist isoforms. Of note, they are more sensitive to hIL-36α and hIL-36γ compared to hIL-36β (see figures). HEK-Blue™ IL-36 cells maintain their responses to other cytokines that signal through NF-κB/AP-1, such as hIL-1β and hTNF-α (see figures). 

Key features:

• Fully functional IL-36 signaling pathway
• Readily assessable NF-κB/AP-1-inducible SEAP reporter activity
• Strong response to human (h) IL-36α and IL-36γ
• Moderate response to hIL-36β
• No response to mouse (m) IL-36α

Applications:

• Detection and quantification of hIL-36 activity
• Screening of anti-IL-36 and anti-IL-36 receptor antibodies
• Screening of small molecule inhibitors of the IL-36 pathway

References:

1. Buhl A-L. & Wenzel J.,  2019. Interleukin-36 in infectious and inflammatory skin diseases. Front Immunol. 10:1162.
2. Zhou L. & Todorovic V.,  2021. Interleukin-36: Structure, Signaling and Function. Adv Exp Med Biol. 21:191-210.

Figures

Human IL-36R mRNA expression in HEK-Blue™ IL-36 cells.
Total mRNA was extracted from ~1x10⁶ HEK-Blue™ parental cells (blue) and HEK-Blue™ IL-36 cells (red). Human IL-36R mRNA was amplified using quantitative RT-qPCR.
Data are represented as the log2 fold change comparing hIL-36R relative expression between the two cell lines.

Dose-response of HEK-Blue™ IL-36 cells to recombinant IL-36.
Cells were stimulated with increasing concentrations of recombinant human IL-36 (hIL-36) and murine IL-36 (mIL-36) isoforms α, β, or γ. After overnight incubation, the NF-κB response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 630 nm is shown as mean ± SEM.

Response of HEK-Blue™ IL-36 cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines: 0.1 ng/ml hIL-36α, 10 ng/ml hIL-36β, 0.1 ng/ml hIL-36γ, 10 ng/ml mIL-36α, 0.03 ng/ml hIL-1β, 1 ng/ml TNF-α, or 1000 U/ml hIFN-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. The optical density (OD) at 630 nm is shown as mean ± SEM.

Specifications

Antibiotic resistance: Blasticidin and  Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin®

Specificity: human IL-36α, IL-36β and IL-36γ

Detection range: 

• 30 pg/ml - 100 ng/ml for hIL-36α and hIL-36γ
• 10 ng/ml - 100 ng/ml for hIL-36β

Quality Control:

• The expression of human IL-36R is assessed by RT-qPCR.
• SEAP reporter activity in response to IL-36 is validated using functional assays.
• The stability for 20 passages following thawing is confirmed.
• These cells are tested for mycoplasma contamination. 
   

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

Contents

• 3-7 x 10⁶ HEK-Blue™ IL-36 cells in a cryovial or shipping flask
• 1 ml Normocin® (50 mg/ml)
• 1 ml Blasticidin™ (10 mg/ml)
• 1 ml Zeocin® (100 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 cytokine interleukin 36 (IL-36) belongs to the IL-1 superfamily. Three isoforms, IL-36α, IL-36β, and IL-36γ, mediate pro-inflammatory functions, while a fourth one, IL-36Ra, acts as an antagonist [1,2]. IL-36 signalization requires the formation of a complex comprised of the IL-36 receptor (IL-36R or IL-1R6) and the IL-1 receptor accessory protein (IL-1RAcP). The binding of agonist ligands to the IL-36R allows the recruitment of IL-1RAcP and the production of pro-inflammatory cytokines and chemokines through the activation of NF-κB and AP-1 [1,2]. The IL-36Ra antagonist inhibits the signaling by binding to IL-36R and preventing the recruitment of IL-1RAcP [1,2]. IL-36  associated immune response mainly takes place in barrier tissues, such as the skin, lungs, and intestines. Dysregulation of IL-36 isoform expression and signaling has been associated with inflammatory diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease [1,2].

1. Buhl A-L. & Wenzel J.,  2019. Interleukin-36 in infectious and inflammatory skin diseases. Front. Immunol. 10(1162). doi: 10.3389/fimmu.2019.01162.
2. Zhou L. & Todorovic V.,  2021. Interleukin-36: Structure, Signaling and Function. Protein Reviews: Volume 21. doi: 10.1007/5584_2020_488.

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