IL-17 Reporter HEK 293 Cells

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IL-17 Reporter Cells

Signaling pathway in HEK-Blue™ IL-17 cells

HEK-Blue™ IL-17 cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human interleukin 17A (IL-17A) and IL-17F, as well as human and murine IL-17E (also known as IL-25), by monitoring the activation of the NF-κB and AP-1 pathways. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the IL-17 pathway.
IL-17 is a family of six closely related cytokines (IL-17A to IL-17F) which have both pro-inflammatory and anti-inflammatory activities [1, 2].

 More details

Cell line description

HEK-Blue™ IL-17 cells were generated by stable transfection with the genes encoding for the human IL-17 receptor (IL-17RA and IL-17RC chains), the ACT1 adaptor molecule, as well as an NF-κB- and AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-17 to its receptor triggers a signaling cascade leading to the activation of NF-κB/AP-1 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-17 cells detect human (h) IL-17A, hIL-17E, hIL-17F, and murine (m) IL-17E. They display little to no response to mIL‑17A and mIL-17F. They do not respond to hIL-17C, mIL-17C. Of note, they are not responsive to hTNF-α and hIL-1β (see figures).

Key features

• Fully functional IL-17 signaling pathway
• Readily assessable NF-κB/AP-1-SEAP reporter activity
• Strong response to hIL-17A, hIL-17F, & h/m IL-17E
• Low to no response to mIL-17A, h/mIL-17C, & mIL-17F
• No response to hTNF-α and hIL-1β

Applications

• Detection and quantification of hIL-17A & hIL-17F activity
• Detection and quantification of h/mIL-17E (IL-25) activity
• Screening of anti-IL-17 or anti-IL-17 receptor antibodies
• Screening and release assay of small molecule inhibitors of the IL-17 pathway

References:

1. Monin L. & Gaffen S.L., 2018. Interleukin 17 family cytokines: signaling mechanisms, biological activities, and therapeutic implications. Cold Spring Harb Perspect Biol. 10(4).
2. Pappu R. et al., 2011. The interleukin-17 cytokine family: critical players in host defence and inflammatory diseases. Immunology. 134: 8-16.

Figures

Dose-response of HEK-Blue™ IL‑17cells to recombinant IL-17 cytokines.

Cells were stimulated with increasing concentrations of recombinant human or murine IL-17A, IL-17E, and IL-17F. After overnight incubation, the NF-κB response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 630 nm. EC50 values are shown as mean ± SD.

Cytokine response profile of HEK-Blue™ IL-17 cells.

Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of hIL‑17A, mIL‑17A, hIL‑17E, mIL‑17E, hIL‑17F, mIL-17F, hIL‑17C, mIL-17C, 1x104 IU/ml of hIFN-α, hIFN-β, 100 ng/ml of hIFN-γ, and 1 ng/ml of hIL‑1β and hTNF-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. Data is shown as mean ± SD.

Specifications

Antibiotic resistance: Blasticidin, Hygromycin, 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®

Specificity: human IL-17A, human IL-17F, and human & mouse IL-17E (IL-25)

Detection range:

• human IL-17A: 1- 100 ng/ml
• human & mouse IL-17E (IL-25): 1- 100 ng/ml
• human IL-17F: 3 - 100 ng/ml

Quality Control:

• SEAP reporter activity in response to IL-17 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 of  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 17 (IL-17) is a family of six closely related cytokines (IL-17A to IL-17F) which have both pro-inflammatory and anti-inflammatory activities. IL-17A and IL-17F, which can form a heterodimer, play an important role in Th17 immunity and are implicated in tumorigenesis and autoimmune diseases, whereas IL-17E (also known as IL-25) appears to promote Th2 immunity [1,2].

IL-17 cytokines exert their biological activities by binding to heterodimeric receptors containing the ubiquitous IL-17RA chain and a second IL-17R(C, B or E) chain. IL-17A and IL-17F bind to the IL-17RA/IL-17RC receptor, IL-17C binds to the IL-17RA/IL-17RE receptor, and IL-17E binds to the IL-17RA/IL-17RB receptor [1, 2]. The activated heterodimeric receptor recruits the Act1 adaptor and induces the TNF receptor-associated factor 6 (TRAF6) ubiquitylation. This triggers a signaling cascade that results in NF-κB and AP-1 activation [1].

1. Monin L. & Gaffen S.L., 2018. Interleukin 17 family cytokines: signaling mechanisms, biological activities, and therapeutic implications. Cold Spring Harb Perspect Biol. 10(4).
2. Pappu R. et al., 2011. The interleukin-17 cytokine family: critical players in host defence and inflammatory diseases. Immunology. 134: 8-16.
 

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