This nuclear receptor assay system utilizes proprietary non-human cells engineered to provide constitutive, high-level expression of the full-length human estrogen receptor 1 (NR3A1), a ligand-dependent transcription factor commonly referred to as ERα. The reporter cells include the luciferase reporter gene functionally linked to an ERα-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in ERα activity. Luciferase gene expression occurs after ligand-bound ERα undergoes nuclear translocation, DNA binding, recruitment and assembly of the co-activators and accessory factors required to form a functional transcription complex, culminating in expression of the target gene. Unlike some other cell-based assay strategies, the readout from The reporter cells demands the same orchestration of all intracellular molecular interactions and events that can be expected to occur in vivo. ERα reporter cells are prepared using the proprietary CryoMite process. This cryo-preservation method yields exceptional cell viability post-thaw, and provides the convenience of immediately dispensing 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, cell titer adjustments, or the pre-incubation of reporter cells prior to assay setup. The principal application of this assay product is in the screening of test samples to quantify functional activities, either agonist or antagonist, that they may exert against the human estrogen receptor. It is an all-inclusive assay system that includes, in addition to ERα reporter cells, two optimized media for use during cell culture and in diluting the test samples, a reference agonist, luciferase detection reagent, a cell culture-ready assay plate, and a detailed protocol.
ERa Reporter Cells: 3 x 0.60 mL; -80°CCell Recovery Medium (CRM): 1 x 10.5 mL; -20°CCompound Screening Medium (CSM): 1 x 35 mL; -20°C17β-Estradiol, 100 uM (in DMSO) (reference agonist for ERa): 1 x 30 μL; -20°CDetection Substrate: 3 x 2.0 mL; -80°CDetection Buffer: 3 x 2.0 mL; -80°CPlate frame: 1; ambientSnap-in, 8-well strips (white, sterile, cell-culture ready): 12; ambient