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TPOR Reporter Assay Kit

Name: TPOR Reporter Assay Kit
SKU: IB2000
Availability: InStock

SIZES SKU

1 x 96-well format assays IB20001

3 x 32 assays in 96-well format IB20001-32

1 x 384-well format assays IB20002

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Specifications
Data
Target Background
Documentation
Overview
Citation
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Thrombopoietin Receptor (TPOR). INDIGO’s TPOR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of Human TPOR. In addition to TPOR Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human TPOR. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

Clear, Reproducible Results
All-Inclusive Assay Systems
Exceptional Cell Viability Post-Thaw
Consistent Results Lot to Lot

Product Specifications

Target Type		Cytokine Receptor
Species		Human
Receptor Form		Native
Assay Mode		Agonist, Antagonist
Kit Components		TPOR Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) TPO, (ref. activator; in DMSO) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Orthologs Available		No
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

TPOR Activation assay. Agonist dose-response analyses were performed according to the protocol provided in the assay Technical Manual. The reference compounds TPO (provided), Eltrombopag, Avatrombopag, and Lusutrombopag (Cayman Chemical, Ann Arbor, MI, USA) were used to confirm assay performance. 200 µl / well of TPOR or ‘Mock’ Reporter Cell suspensions were dispensed into the 96-well assay plate, then incubated for 4 hours. The reference agonists were further diluted using CSM to produce treatment media at the desired assay concentrations. Pre-culture media were discarded from the assay wells and 200 µl per well of the prepared treatment media were dispensed (n = 3/conc.), including ‘untreated’ control wells. ‘Mock’ reporter cells, which contain the STAT5-Luc reporter gene, but lack expression of TPOR, were similarly treated with TPO. Following a 22 hr incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD), percent coefficient of variation (%CV), Fold-Activation and Z’ were determined for each treatment concentration. Non-linear regression analyses of Fold Activation vs. Log10[Compounds, ng/ml] and EC50 determination was performed using GraphPad Prism software. The absence of signal in TPO-treated ‘Mock’ reporter cells confirms that the response observed is specific to TPOR function.

Target Background

This assay utilizes proprietary human cells that provide constitutive expression fo the Human Thrombopoietin Receptor (TPOR).

TPOR, also known as MPL or CD110, is a member of the type I cytokine receptor superfamily. Thrombopoietin (TPO), the physiological activator of TPOR, is a glycoprotein that regulates thrombopoiesis, the process of platelet production, as well as hematopoietic stem cell maintenance. TPO, which is constitutively produced in the liver with some contribution by the kidneys, binds to and activates the cell-surface TPOR to initiate signal transduction through several different pathways, including JAK/STAT, MAPK, and PI3K pathways.

JAK2 dependent phosphorylation and activation of the transcription factor STAT5 is a prominent outcome of TPOR activation, and it is the signaling pathway exploited by the reporter cells included in this assay. Specifically, INDIGO’s Reporter Cells contain the luciferase reporter gene functionally linked to an engineered minimal promoter sequence with upstream tandem STAT5 genetic response element (GRE) sequences. Thrombopoietin activates TPOR in a dose-dependent manner, thereby triggering the JAK2/STAT signal transduction cascade. Activated STAT5 binds to its consensus GREs to initiate the formation of a complete transduction complex that drives expression of the Luc reporter gene. Therefore, quantifying changes in luciferase activity from peptide-, drug-, or antibody-treated reporter cells relative to that of “untreated” cells provides a sensitive surrogate measure of the changes in the activity of TPOR.

TPOR has been targeted successfully in the clinic to treat conditions such as chronic immune thrombocytopenia, severe aplastic anemia, thrombocytopenia in hepatitis C patients undergoing interferon-based treatments, and thrombocytopenia in adults with chronic liver disease who will be undergoing a medical procedure. Examples of FDA-approved drugs that target TPOR include Romiplostim, Eltrombopag, Avatrombopag, ad Lusutrombopag. Accordingly, the primary application of this reporter assay is test compound as a TPOR agonist or TPOR antagonist.