IL-36 Reporter HEK 293 Cells

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IL-36 responsive NF-κB/AP1-SEAP reporter assay

Signaling pathway in HEK-Blue™ IL-36 cells

HEK-Blue™ IL-36 cells are designed to monitor human IL-36-induced NF-κB/AP1 stimulation or inhibition. This colorimetric cytokine bioassay can be used to screen activatory or inhibitory molecules, such as engineered cytokines and neutralizing antibodies, respectively.

IL-36 reporter cells respond to recombinant human IL-36α, IL-36β, and  IL-36γ, but not to mouse IL-36 (see figures). Their reliable and consistent performance makes them suitable for release assays of activatory and inhibitory molecules such as Imsidolimab, a monoclonal antibody that targets the IL-36 receptor (IL-36R) and prevents IL-36 signaling (see figures).

Key features

• Readily assessable NF-κB/AP-1-inducible SEAP reporter activity
• Convenient readout using QUANTI-Blue™ Solution
• Strong response to human (h) IL-36α and IL-36γ
• No response to mouse (m) IL-36α
• Stability guaranteed for 20 passages 

Applications

• Therapeutic development
• Drug screening
• Release assay

The interleukin-36 (IL-36) belongs to the IL-1 superfamily and comprises multiple isoforms, whereas IL-36α, IL-36β, and IL-36γ mediate pro-inflammatory functions, while a fourth one, IL-36Ra, acts as an antagonist. Dysregulation of IL-36 isoform expression and signaling has been associated with inflammatory diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease.

 More details

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 650 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ IL-36 cell response using Imsidolimab biosimilar. Increasing concentrations of Anti-hIL-36R-hIgG4 (S228P) (0.1 ng/ml - 10 µg/ml) were incubated with HEK‑Blue™ IL-36 cells for 1 hour before the addition of recombinant human IL-36α (30 pg/ml). After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown as the percentage of activity (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 650 nm is shown as mean ± SEM.

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: IL-36α, IL-36β, IL-36γ

Specificity: Human

Reporter gene: SEAP

Antibiotic resistance: Blasticidin and  Zeocin®

Detection range: 30 pg/ml - 100 ng/ml (hIL-36α and hIL-36γ), 10 ng/ml - 100 ng/ml (IL-36β)

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-36 Cells (hkb-il36r)

• 1 vial containing 3-7 x 10⁶ cells
• 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)
   

HEK-Blue™ IL-36 vial (hkb-il36r-av)

• 1 vial containing 3-7 x 10⁶ cells

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

Notification:  Reference #hkb-il36r-av can only be ordered together with reference #hkb-il36r.

Details

Cell line description

HEK-Blue™ IL-36 cells were generated by stable transfection with the genes encoding for the human IL-36 heterodimeric receptor, comprising the IL-1R6 (aka IL-36R) and IL-1R3 (aka IL-1RAcP) subunits, 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.
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). 

IL-36 background

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 two subunits, IL-1R6 (aka IL-36R) and IL-1R3 (aka IL-1RAcP, IL-1 receptor accessory protein). 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].

​References:

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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