TPO Reporter HEK 293 Cells

HEK-Blue™ TPO Cells signaling pathway

TPO Reporter 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.  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 overexpression of the genes encoding for the human TPO receptor (TPO-R; also known as c-Mpl), human STAT5b, and a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. STAT5-dependent SEAP activity is readily assessable in the supernatant using QUANTI-Blue™ Solution, a detection reagent. Of note, as HEK293 cells endogenously express the receptors for type I and type III interferons (IFNs), these cells also respond to IFN-α/β and IFN-γ (see figures). 

Features of HEK-Blue™ TPO cells:

• Fully functional TPO signaling pathway
• Readily assessable STAT5-inducible SEAP reporter activity
• The stability for 20 passages has been verified
• Functionally tested and guaranteed mycoplasma-free

Applications of HEK-Blue™ TPO cells:

• Detection of human TPO (3 - 100 ng/ml), and  murine TPO (0.1 -10 ng/ml)
• Screening of anti-TPO and anti-TPO receptor antibodies

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, 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: human & murine TPO

Detection range: 

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

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 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™ TPO 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

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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