G-CSF Reporter HEK 293 Cells

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Granulocyte Colony-Stimulating Factor Reporter Cells

Signaling pathway in HEK-Blue™ G-CSF cells

HEK-Blue™ G-CSF cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive granulocyte colony-stimulating factor (G-CSF) by monitoring the activation of the JAK/STAT3 pathway. They can also be used for screening antibodies or small molecule inhibitors targeting the G-CSF pathway.

G-CSF is a secreted cytokine and hematopoietic growth factor. It regulates the differentiation, proliferation, and function of neutrophils [1].

 More details

Cell line description

HEK-Blue™ G-CSF cells were generated by stable transfection with the genes encoding for the human CSF receptor (CSFR, aka CD114), human STAT3c, and a STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of G-CSF to its receptor triggers a signaling cascade leading to the activation of STAT3 and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ G-CSF cells detect human and murine G-CSF (see figures). Of note, these cells also respond, to a weaker extent, to two other human STAT3-signaling cytokines, IL-6 and IL-27, as they endogenously express the gp130 co-receptor shared by these cytokine receptors. However, they do not respond to human type I IFNs (IFN-α/IFN-β) (see figures).

Key features

• Fully functional G-CSF signaling pathway
• Readily assessable STAT3-inducible SEAP reporter activity
• Strong response to human (h) G-CSF and mouse (m) C-CSF
• Weak response to IL-6 and IL-27
• No response to human type I IFNs

Applications

• Detection and quantification of human and murine G-CSF activity
• Screening of anti-G-CSF and anti-GCSFR antibodies
• Screening of small molecule inhibitors of the G-CSF pathway

Reference:

1. Martin KR, et al., 2021. G-CSF — A double-edged sword in neutrophil-mediated immunity. Sem. Immunol. 54:101516.

Figures

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

Dose-response of HEK-Blue™ G-CSF cells to recombinant G-CSF. Cells were stimulated with increasing concentrations of recombinant human G-CSF (hG-CSF) and murine G-CSF (mG-CSF). After overnight incubation, the STAT3 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™ G-CSF cellular response using a neutralizing antibody against human G-CSF. An increasing concentration of a neutralizing anti-hG-CSF antibody was incubated with recombinant human G-CSF (0.1 ng/ml) for 2 hours before the addition of HEK-Blue™ G-CSF cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™. Data are shown as a percentage (%) of activity.

Response of HEK-Blue™G-CSF cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of hG-CSF, mG-CSF, hGM-CSF, mGM-CSF, hIL-6, hIL-27, hIFN-γ, and 1000 U/ml hIFN-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™. The OD at 630 nm is shown as mean ± SEM.

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, 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: Detects human and mouse G-CSF

Detection range:

• Detection range for human G-CSF: 300 pg/ml - 100 ng/ml
• Detection range for murine G-CSF: 300 pg/ml - 100 ng/ml

Quality Control:

• SEAP reporter activity in response toG-CSF is validated using functional assays.
• The expression of human GCSFR is confirmed by flow cytometry.
• 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
• 1 ml Normocin® (50 mg/ml)
• 2 x 1 ml of HEK-Blue Selection (250X)
• 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)

Details

The granulocyte colony-stimulating factor (G-CSF) is a secreted cytokine and hematopoietic growth factor that belongs to the Type I/II cytokine receptor family. G-CSF regulates the differentiation, proliferation, and function of neutrophils [1]. It exerts its biological functions through the formation of a tetramer consisting of two G-CSF molecules and the homodimeric G-CSF receptor (GCSFR, aka CD114). The binding of G-CSF to its receptor triggers activation of the JAK/STAT signaling pathway [1,2]. STAT1, STAT3, and STAT5 are involved in this activation step and the induction of cellular proliferation [1,2]. Studies in humans and mice indicate that while G-CSF contributes to host defense against pathogens, it can also have detrimental effects by promoting inflammatory diseases and cancer [1,2].

​References:

1. Martin KR., et al., 2021. G-CSF — A double-edged sword in neutrophil-mediated immunity. Sem Immunol. 54:101516.
2. Park, DS., et al., 2022. A review of granulocyte colony-stimulating factor receptor signaling and regulation with implications for cancer. Front Oncol. DOI: 10.3389/fonc.2022.932608.

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