Nuclear Factor of Activated T cells (NFAT)

This Nuclear Factor of Activated T cells (NFAT) kit product is an all-inclusive assay system that includes addition to NFAT Reporter Cells, two optimized media for use during cells culture and in diluting test samples, the calcineurin-NFAT pathway activator Ionomycin, Luciferase Detection Reagent, and a cell culture-ready assay plate.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 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.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. The five members of the Nuclear Factor of Activated T cells (NFAT1-5) family were initially identified as key regulators of genes involved in the activation, proliferation, differentiation, and apoptosis of cells, most notably the T cells and B cells of the immune system.Inactive NFAT resides in the cytoplasm in a multi-phosphorylated form. Phospho-NFAT is converted to its active form through the activation of calcineurin, a calcium-dependent phosphatase. Any physiological event that drives the influx of extra-cellular Ca+2, or depletes internal Ca+2 stores within the endoplasmic reticula, results in the Ca+2-activation of calcineurin and its subsequent dephosphorylation of phospho-NFAT. Activated NFAT translocates to the nucleus where it binds to specific genetic response element (GRE) sequences within the promoter / enhancer region of target genes. Functional transcription complexes form via cooperative association with other transcription factors, most notably AP-1.Importantly, dysregulation of the calcineurin-NFAT pathway is strongly associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, thereby making it an important therapeutic target for small molecule drug development.INDIGO's Reporter Cells contain an engineered luciferase reporter gene functionally linked to tandem NFAT/AP-1 GRE sequences positioned immediately upstream of a minimal promoter. Activated NFAT will bind to its corresponding GREs to initiate the formation of a complete transcription complex that drives Luc gene expression. Thus, quantifying changes in luciferase activity in the treated reporter cells provides a sensitive surrogate measure of changes in NFAT activity. Accordingly, the principal application of this reporter assay is in the screening of test compounds to quantify any functional activities, either activating or inhibitory, that they may exert against the calcineurin-NFAT signal transduction pathway.