The Mouse Constitutive Androstane Receptor (nr1i3) Assay is a cell-based genetic reporter assay that utilizes proprietary non-human mammalian cells engineered to provide constitutive, high-level expression of a ligand-dependent transcription factor commonly referred to as mCAR. These reporter cells utilize a modified version of mCAR in which the native N-terminal DNA binding domain (DBD) has been replaced with that of the GAL4-DBD. The mCAR ligand binding domain (LBD) is unaltered and fully functional. The reporter cells also incorporate a luciferase cDNA functionally linked to the GAL4-upstream activation sequence (UAS). Thus, quantifying expressed luciferase activity provides a sensitive surrogate measure of changes in mCAR activity resulting from direct interaction between a treatment compound and the nuclear receptor. Because this assay system expresses the [GAL4-DBD + mCAR LBD] hybrid receptor, the activity of modulators that act through indirect mechanisms (such as those that alter the phosphorylation status of the native N-terminal amino acid sequence of the CARs) may be dampened or go undetected.Contrary to its name, mouse CAR is not constitutively active, rather, it exhibits ligand-dependent activation. The primary application of this reporter assay system is in the screening of test compounds to quantify any functional activity, either agonist or antagonist, that they may exert on mouse CAR.mCAR 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.TheMouse CAR assay kit is an all-inclusive system. In addition to mCAR Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user"s test samples, a reference agonist (TCPOBOP), Luciferase Detection Reagent, and a cell culture-ready assay plate.
▪ mCAR Reporter Cells: 1 x 2.0 mL; -80°C ▪ Cell Recovery Medium (CRM): 1 x 10.5 mL; -20°C▪ Compound Screening Medium (CSM): 1 x 35 mL; -20°C ▪ TCPOBOP*, 10 mM (in DMSO) (reference agonist for mCAR): 1 x 30 µL; -20°C▪ Detection Substrate: 1 x 6.0 mL; -80°C▪ Detection Buffer: 1 x 6.0 mL; -20°C ▪ 96-well assay plate (white, sterile, cell-culture ready): 1; ambient