Description
This assay utilizes proprietary human cells that provide constitutive expression of the Human Granulocyte Colony-Stimulating Factor Receptor (G-CSFR). Binding of G-CSF to its receptor G-CSFR, is known to initiate signal transduction through JAK/STAT, PI3K/AKT and other pathways including those that lead to the activation of ERK1/2. The activation of these various pathways may culminate in the activation of cytosolic targets, or in the activation of specific transcription factors and the induction of their respective target genes. JAK dependent phosphorylation and activation of the transcription factor STAT5 is a prominent outcome of G-CSFR activation, and it is the signaling pathway exploited by the reporter cells included in this assay kit. 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. G-CSF activates the G-CSFR in a dose-dependent manner, thereby triggering the JAK/STAT signal transduction cascade. Activated STAT5 binds to its consensus GREs to initiate the formation of a complete transcription 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, dosedependent surrogate measure of changes in the activity of G-CSFR. INDIGO’s Reporter Cells are transiently transfected and prepared as frozen stocks using a proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations or cell titer adjustments prior to assay setup. This assay kit provides the convenience of an all-inclusive cell-based reporter assay system for G-CSFR signaling. In addition to Reporter Cells, included are an optimized culture medium for use in reviving the cryopreserved cells, a culture medium for use in preparing test sample treatments, the physiological activator G-CSF as a positive control, Luciferase Detection Reagents, and a cell culture-ready assay plate. [INDIGO Catalog Nos. IB30101 | IB30102]WARNING This product is not for human or veterinary use.
