Wnt signaling via beta - catenin plays a central role in development and homeostasis. This pathway is invariably disrupted in colorectal tumors and commonly affected by mutation in other cancers. Binding of the Wnt protein to the Frizzled transmembrane receptors initiates a signaling cascade that activates the Dishevelled protein, which then inhibits the serine/threonine kinase Glycogen Synthase - 3 Beta (GSK - 3B). This signal leads to functional inactivation and dissociation of a multiprotein beta - catenin destruction complex made up of the tumor suppressor protein Adenomatous Polyposis Coli (APC), GSK - 3B, and a scaffold of Axin. This results in dephosphorylation and dissociation of beta - catenin. Unphosphorylated beta - catenin is stabilized and accumulates in the cytoplasm of the cell. Beta - catenin then associates with the Lymphoid Enhancer Factor (LEF)/T - Cell Factor (TCF) family of transcription factors in the nucleus, leading to transcription and expression of target genes, such as c - Myc, c - jun, Fra, and cyclin D1. The CellSensor® LEF/TCF - bla FreeStyle™ 293 Cell Line contains a beta - lactamase reporter gene under control of the LEF/TCF stably integrated into FreeStyle™ 293 cells. This cell line is validated for EC50 and Z' - factor under optimized conditions using mWnt3a. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. CellSensor® LEF/TCF - bla FreeStyle™ 293F cells were stimulated with mWnt3a over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios are shown plotted against the indicated concentrations of mWnt3a. Academic and non-profit customers, please inquire for special pricing.