TPO Reporter HEK 293 Cells

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Thrombopoietin reporter cells

Signaling pathway in HEK-Blue™ TPO cells

HEK-Blue™ TPO cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive thrombopoietin (TPO) by monitoring the activation of the JAK2/STAT5 pathway. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the TPO pathway. 

TPO is a key hematopoietic growth factor that plays a vital role in the regulation of megakaryocytopoiesis and the maintenance of hematopoietic stem cells (HSCs) [1].

 More details

Cell line description

HEK-Blue™ TPO cells were generated by stable transfection with the genes encoding for the human TPO receptor (TPO-R, aka CD110), human STAT5b, and a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of TPO 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™ TPO cells detect human and murine TPO (see figures). Of note, as HEK293 cells endogenously express the receptors for type I and type II interferons (IFNs), these cells also respond to IFN-α/β and IFN-γ (see figures). 

Key features

• Fully functional TPO signaling pathway
• Readily assessable STAT5-inducible SEAP reporter activity
• Strong response to human (h) and murine (m) TPO
• Response to hIFN-α, hIFN-β, and IFN-γ

Applications

• Detection and quantification of human and murine TPO activity
• Screening of anti-TPO and anti-TPO receptor antibodies
• Screening of small molecule inhibitors of the TPO pathway

Reference:

1. Grozovsky R. et al., 2015. Novel mechanisms of platelet clearance and thrombopoietin regulation. Curr Opin Hematol 22(5):445-51.

Figures

Surface expression of human TPO-R by HEK-Blue™ TPO cells.
~2 x 10⁵ HEK‑Blue™ TPO cells were incubated with either a PE‑conjugated Anti‑hTPO-R mAb or a PE-conjugated mouse IgG2b isotype control for 1 hour. Cell surface staining was analyzed by flow cytometry.

Figure 1. Dose-response of HEK-Blue™ TPO cells to recombinant TPO. Cells were stimulated with increasing concentrations of recombinant human TPO (hTPO) and murine TPO (mTPO). After overnight incubation, the STAT5 response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 630 nm. Data are shown as mean ± SEM.

Figure 2. Response of HEK-Blue™ TPO cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines:
10 ng/ml of hTPO, mTPO,  hIL‑2, hIL-15, hIFN-γ, or 30 U/ml hIFN-α or hIFN-β. 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, 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 TPO and mouse TPO

Detection range: 

• Detection range for human TPO: 3 ng/ml - 100 ng/ml
• Detection range for murine TPO: 0.1 ng/ml - 10 ng/ml

Quality control:

• STAT5-dependent SEAP reporter activity in response to human and murine TPO has been validated.
• The expression of human STAT5b has been confirmed by RT-qPCR.
• The cell surface expression of human TPO-R in this cell line has been validated using flow cytometry.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

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 and some areas in Asia)

Details

TPO is the chief cytokine that regulates megakaryocyte production, signaling through its receptor (TPO-R; also known as c-Mpl). It also plays a vital role in the maintenance of hematopoietic stem cells (HSCs) [1]. TPO promotes the proliferation and differentiation of megakaryocytes and their specialized progenitors. Additionally, TPO drives platelet production and activity. The number of circulating platelets must be tightly controlled to avoid spontaneous bleeding or arterial occlusion and organ damage. Abnormal levels of TPO or mutations in its receptor are associated with blood disorders, such as thrombocytosis and thrombocytopenia. TPO is produced principally in the liver and is found in peripheral blood. TPO acts by binding to the extracellular portion of the cell surface TPO-R. Upon binding to its receptor, TPO activates JAK2/STAT5 signaling which initiates transcriptional elements and the expression of TPO-inducible genes [2, 3]. 

1. Grozovsky R. et al., 2015. Novel mechanisms of platelet clearance and thrombopoietin regulation. Curr Opin Hematol 22(5):445-51.
2. Ghanima W. et al., 2019. Thrombopoietin receptor agonists: ten years later. Haematologica. 104(6):1112-23.
3. Hitchcock I. & Kaushansky K., 2014. Thrombopoietin from beginning to end. Br J Haematol. 165(2):259-68.
 

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