TPOR Reporter Assay System [Human]

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 to screen test materials for any functional activity, either agonistic or inhibitory, that they may exert against human TPOR. Protein function: Receptor for thrombopoietin that acts as a primary regulator of megakaryopoiesis and platelet production. May represent a regulatory molecule specific for TPO-R-dependent immune responses. [The UniProt Consortium] 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. Assay systems are all inclusive, providing reporter cells, optimized growth media, media for diluting test compounds, a positive-control agonist, luciferase detection reagent, a cell culture-ready assay plate, and a detailed protocol. All kits are shipped on dry ice. Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's proprietary CryoMite(TM) process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. INDIGO's assay kits provide the convenience of an all-inclusive cell-based assay system. In addition to TPOR Reporter Cells, included are an optimized culture medium for use in reviving the cryopreserved cells and for diluting test samples, the physiological activator Thrombopoietin, Luciferase Detection Reagents, and a cell culture-ready assay plate.