Thrombopoietin Receptor (TPOR)
|Product Family||Product Number||Product Description||Technical Manual|
|IB20001-32||Human TPOR Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB20001||Human TPOR Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB20002||Human TPOR Reporter Assay System, 1 x 384-well format assays|
Thrombopoietin Receptor Assay Kit
This Thrombopoietin Receptor (TPOR) reporter assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition to the TPOR Reporter Cells, two optimized media for use in recovering the cryopreserved cells and for diluting test samples. Also included is the reference agonist EGF, Luciferase Detection Reagents, and a cell culture-ready assay plate.
TPOR Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments. .
INDIGO’s TPOR reporter assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
TPOR reporter assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Thrombopoietin Receptor Assay Services
The primary application of INDIGO's cell-based TPOR assays are to quantitatively assess the bioactivity of a test compound as a TPOR agonist or TPOR antagonist. Service assays include a positive control reference compound and 'vehicle' control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online "Request a Quote" form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Thrombopoietin Receptor Assay 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.
Synonyms:Thrombopoietin Receptor, TPOR, MPL, CD110
Thrombopoietin Receptor Assay 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.