GM-CSF Reporter HEK 293 Cells

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HEK-Blue™ GM-CSF Cells signaling pathway

Granulocyte-Macrophage Colony-Stimulating Factor Reporter Cells

HEK-Blue™ GM-CSF cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive granulocyte-macrophage colony-stimulating factor (GM-CSF, aka CSF2) by monitoring the activation of the JAK2/STAT5 pathway.  Although originally identified as a hematopoietic growth factor, this cytokine is now regarded as a pleiotropic regulator of inflammation in the responses to pathogens, autoimmune diseases, and cancer [1, 2].

More details

Cell line description:

HEK-Blue™ GM-CSF cells were generated by stable transfection with the genes encoding for human GMRα (CD116), GMRβ (CD131), STAT5, as well as a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of GM-CSF to its receptor triggers a signaling cascade leading to the activation of STAT5 and the subsequent production of SEAP. This can be readily assessable in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ GM-CSF cells detect human but not murine GM-CSF (see figures). Of note, these cells are not responsive to human IL-6, IL-27, and type I IFN (see figures).

Key features:

• Fully functional GM-CSF signaling pathway
• Readily assessable STAT5-inducible SEAP reporter activity

Applications:

• Detection of human GM-CSF
• No detection of murine GM-CSF
• Screening of anti-GM-CSF and anti-GMRα 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. Zhan Y. et al., 2019. The Pleiotropic Effects of the GM-CSF Rheostat on Myeloid Cell Differentiation and Function: More Than a Numbers Game. Front Immunol. 102679.
3. Hamilton J.A., 2020. GM-CSF in inflammation. J. Exp . Med. 217(1):e20190945.

Figures

Validation of the expression of human GMRα by HEK-Blue™ GM-CSF cells.
5 x 10⁵ cells were incubated with either an APC-conjugated isotype control (blue) or an APC-conjugated Anti-GMRα mAb (red) for 1 hour. The binding affinity was then measured using flow cytometry.

Dose-response of HEK-Blue™ GM-CSF cells to recombinant GM-CSF.
Cells were stimulated with increasing concentrations of recombinant human GM-CSF (hGM-CSF) and murine GM-CSF (mGM-CSF). 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.

Response of HEK-Blue™ GM-CSF cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines: 0.1 ng/ml of hGM-CSF, 10 ng/ml mGM-CSF, hIL‑6, hIL-27, or 1000 U/ml hIFN-α. 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, and Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS; 30 min at 56°C), 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Specificity: human GM-CSF

Detection range:  5 pg/ml - 100 ng/ml for human GM-CSF

Quality Control:

• STAT5-dependent SEAP reporter activity in response to human GM-CSF has been validated.
• The cell surface expression of human GMRα  (CD116) in this cell line has been validated using fluorescence-activated cell sorting (FACS).
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ HEK-Blue™ GM-CSF cells in a cryovial or shipping flask
• 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 and some areas in Asia)

Details

The granulocyte-macrophage colony-stimulating factor (GM-CSF, aka CSF2) belongs to the β-common chain cytokine family [1]. Although originally identified as a hematopoietic growth factor, this cytokine is now regarded as a pleiotropic regulator of inflammation in the responses to pathogens, autoimmune diseases, and cancer [1, 2]. GM-CSF signalization requires a multimeric structure comprising four α chains (GMRα, aka CD116), four β chains (GMRβ aka CD131), and four cytokines. This 12-protein complex allows the juxtaposition of the intracellular Janus kinase 2 (JAK2) and activation of the signal transducer and activator of transcription 5 (STAT5) [1]. Other signaling pathways include ERK, NF-κB, and AKT pathways [2, 3]. 

The understanding of cellular and molecular mechanisms whereby GM-CSF exerts its varied functions is key for the development of therapeutic strategies (e.g. cancer vaccines, blocking antibodies) [1]. 

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. Zhan Y. et al., 2019. The Pleiotropic Effects of the GM-CSF Rheostat on Myeloid Cell Differentiation and Function: More Than a Numbers Game. Front Immunol. 102679.
3. Hamilton J.A., 2020. GM-CSF in inflammation. J. Exp . Med. 217(1):e20190945.

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