IL-9 Reporter HEK 293 Cells

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Interleukin-9 Reporter Cells

Signaling pathway in HEK-Blue™ IL-9 cells

HEK-Blue™ IL-9 cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive interleukin-9 (IL-9) by monitoring the activation of the JAK/STAT5 pathway.  In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the IL-9 pathway.

Originally identified as a growth factor of T cells and mast cells, IL-9 is now regarded as a multifunctional cytokine that regulates the function of many different cell types and plays an important role in immunity and immune pathogenesis [1-3].

 More details

Cell line description

HEK-Blue™ IL-9 cells were generated by stable transfection with the genes encoding the human IL-9 receptor (IL-9Rα and IL-2Rγ chains), human JAK3, human STAT5b, and a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-9 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-9 cells detect human and murine IL-9 (see figures). Of note, HEK‑Blue™ IL-9 cells also exhibit a moderate response to human IFN-γ but do not respond to human IFN-α (see figures).

Key features

• Fully functional IL-9 signaling pathway
• Readily assessable STAT5-inducible SEAP reporter activity
• Strong response to human (h) and mouse (m) IL-9

Applications

• Detection and quantification of hIL-9 and mIL-9 activity
• Screening of anti-IL-9 and anti-IL-9 receptor antibodies
• Screening of small molecule inhibitors of the IL-9 pathway

References:

1. Ritobrata G. & Kaplan M.H., 2011. A brief history of IL-9. J Immunol. 186(6):3283-8.
2. Chakraborty S. et al., 2019. An update on interleukin-9: From its cellular source and signal transduction to its role in immunopathogenesis. Int J Mol Sci. 20(9):2113.
3. Patrussi L. et al., 2021. Interleukin (IL)-9 supports the tumor-promoting environment of chronic lymphocytic leukemia. Cancers (Basel). 13(24):6301.

Figures

Validation of the expression of human IL-9Rα by HEK-Blue™ IL-9 cells.
5 x 10⁵ cells were incubated with either a PE-conjugated isotype control (blue) or a PE-conjugated Anti-IL-9Rα mAb (red) for 30 minutes. The binding affinity was then measured using flow cytometry.

Dose-response of HEK-Blue™ IL-9 cells to recombinant IL-9.
Cells were stimulated with increasing concentrations of recombinant human IL-9 (hIL-9) and murine IL-9 (mIL-9). 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-9 cellular response using a neutralizing antibody against hIL-9.
The antibody was incubated with hIL-9 (0.3 ng/ml) for 2 hours prior to the addition of HEK-Blue™ IL-9 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-9 cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines: 1 ng/ml of hIL-9 or mIL-9, and 100 ng/ml hIL-2, hIL-4, hIL‑6, hIL-7, mIL-7, hIL‑15, hIL-21, or hIL-27, and 1000 U/ml hIFN-α, 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 B, Puromycin, 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®

Specificity: Detects human IL-9 and mouse IL-9

Detection range:

• Detection range for human IL-9: 100 pg - 100 ng/ml
• Detection range for mouse IL-9: 30 pg - 100 ng/ml

Quality Control:

• SEAP reporter activity in response to IL-9 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 3-7 x 10⁶ cells
• 2 x 1 ml HEK-Blue™ Selection (250x concentrate)
• 1 ml of Puromycin (10 mg/ml)
• 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 9 (IL-9) is a secreted cytokine produced primarily by T helper 2 (Th2) cells. It supports the growth of activated T cell subsets and promotes mast cell proliferation and differentiation [1, 2]. In addition, IL-9 has a direct effect on epithelial cell function stimulating the production of mucin and T cell chemoattractants [2, 3].
IL-9 signals through the heterodimeric cell surface IL-9 receptor (IL-9R) consisting of IL-9Rα (also called CD129) and IL-2Rγ (also called the common γ-chain or CD132). The binding of IL-9 to its receptor triggers three main signaling pathways: JAK/STAT, MAPK, and PI3K [1, 2]. IL-9 is an interesting therapeutic target as it plays a critical role in immunity to parasitic infections, the pathogenesis of cancer and immuno-pathological conditions such as asthma [5, 6].  

1. Ritobrata G. & Kaplan M.H., 2011. A brief history of IL-9. J Immunol. 186(6):3283-8.
2. Chakraborty S. et al., 2019. An update on interleukin-9: From its cellular source and signal transduction to its role in immunopathogenesis. Int J Mol Sci. 20(9):2113.
3. Longphre M. et al., 1999. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest . 104(10):1375-82.
4. Little F.F. et al., 2001. Il-9 stimulates release of chemotactic factors from human bronchial epithelial cells.Am J Respir Cell Mol Biol . 25(3):347-52.
5. Silva N.S.L. et al., 2021. Interleukin-9 in immunopathology of Trypanosoma cruzi experimental infection. Front Cell Infect Microbiol. 11:756521. 
6. Patrussi L. et al., 2021. Interleukin (IL)-9 supports the tumor-promoting environment of chronic lymphocytic leukemia. Cancers (Basel). 13(24):6301.

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