IL-5 Reporter HEK 293 Cells

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HEK-Blue™ IL-5 Cells signaling pathway

Interleukin-5 Reporter Cells

HEK-Blue™ IL-5 cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive interleukin-5 (IL-5) by monitoring the activation of the JAK/STAT5 pathway. IL-5 is a homodimeric cytokine that functions principally in the eosinophil arm of the Th2 response [1-3].

 More details

Cell line description:

HEK-Blue™ IL-5 cells were generated by stable transfection with the genes encoding the human IL-5 receptor (IL-5Rα and IL-5RB chains), human STAT5b, and a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-5 to its receptor triggers a signaling cascade leading to the activation of STAT5 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-5 cells detect human (h) and murine (m) IL-5 (see figures). Of note, these reporter cells also respond to hIFN-γ, but not to human type I IFNs (IFN-α/IFN-β) (see figures).

Key features:

• Fully functional IL-5 signaling pathway
• Readily assessable STAT5-inducible SEAP reporter activity

Applications:

• Detection of human and murine IL-5
• Screening of anti-IL-5 and anti-IL-5R antibodies

References:

1. Dougan M. et al., 2019. GM-CSF, IL-3, and IL-5 family of cytokines: regulators of inflammation. Immunity. 50(4):796-811.
2. Morris R. et al., 2018. The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Sci. 27(12):1984-2009.
3. Yanagibashi T. et al., 2017. Allergic diseases: From bench to clinic - Contribution of the discovery of interleukin-5. Cytokine. 98:59-70.

Figures

Validation of the expression of human IL-5Rα by HEK-Blue™ IL-5 cells.
5 x 10⁵ cells were incubated with either a PE-conjugated isotype control (blue) or a PE-conjugated Anti-IL-5Rα mAb (red) for 30 minutes. The binding affinity was then measured using flow cytometry.

Dose-response of HEK-Blue™ IL-5 cells to recombinant IL-5.
Cells were stimulated with increasing concentrations of recombinant human IL-5 (hIL-5) and murine IL-5 (mIL-5). After overnight incubation, the STAT5 response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK‑Blue™ IL-5 cellular response using a neutralizing antibody against IL-5.
The antibody was incubated with hIL-5 (0.1 ng/ml) for 2 hours prior to the addition of HEK-Blue™ IL-5 cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown as a percentage (%) of activity

Response of HEK-Blue™ IL-5 cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines: 0.3 ng/ml of hIL-5 or mIL-5, and 100 ng/ml hIL-3, hIL‑6, hIL-27, or hGM-CSF, and 1000 U/ml hIFN-α or hIFN-β, and 10 ng/ml hIFN-γ. 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, Hygromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 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 murine interleukin 5

Detects human and murine IL-5: 30 pg - 100 ng/ml

Contents

• 1 vial containing 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 5 (IL-5) is a secreted pro-inflammatory cytokine that specifically induces the differentiation of eosinophils, which play important roles in host defense as well as in pathologies such as allergies and asthma [1]. It also stimulates B cell growth and increases immunoglobulin secretion. Homodimeric IL-5 signals through the heterodimeric cell surface IL-5 receptor (IL‑5R) consisting of IL-5Rα (also called CD125) and IL-5RB, (also called the cytokine receptor common subunit beta, CSF2RB or CD131).
The binding of IL-5 to its receptor triggers three main signaling pathways: JAK/STAT, PI3K, and MAPK/ERK [1‑2]. Of note, monomeric IL-5 has no activity [2]. The pivotal role of IL-5 in eosinophil differentiation makes it an attractive target in eosinophilic conditions such as asthma [3].

1. Dougan M. et al., 2019. GM-CSF, IL-3, and IL-5 family of cytokines: regulators of inflammation. Immunity. 50(4):796-811.
2. Morris R. et al., 2018. The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Sci. 27(12):1984-2009.
3. Yanagibashi T. et al., 2017. Allergic diseases: From bench to clinic - Contribution of the discovery of interleukin-5. Cytokine. 98:59-70.

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