This nuclear receptor assay system utilizes proprietary non-human mammalian cells engineered to provide constitutive, high-level expression of human constitutive androstane receptor, isoform 3 (NR1I3), a ligand-dependent transcription factor commonly referred to as CAR3. These reporter cells utilize a modified version of human CAR3 in which the native N-terminal DNA binding domain (DBD) has been replaced with that of the GAL4-DBD. The human CAR3 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 CAR3 activity resulting from direct interaction between a treatment compound and the nuclear receptor. Because this assay system expresses the [GAL4-DBD + hCAR3 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, human CAR3 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 human CAR3. Reporter cells are prepared using the proprietary CryoMite process. This cryopreservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments. The nuclear receptor reporter assays are all-inclusive cell-based assay systems. In addition to CAR3 reporter cells, this kit provides two optimized media for use during cell culture and in diluting the user"s test samples, a positive-control agonist, luciferase detection reagent, and a cell culture-ready assay plate.
▪ CAR3 Reporter Cell:s 3 x 0.60 mL; -80°C ▪ Cell Recovery Medium (CRM): 1 x 10.5 mL; -20°C▪ Compound Screening Medium (CSM): 1 x 35 mL; -20°C▪ CITCO*, 4.0 mM (in DMSO) (reference agonist for CAR3): 1 x 30 µL; -20°C ▪ Detection Substrate: 3 x 2.0 mL; -80°C▪ Detection Buffer: 3 x 2.0 mL; -80°C▪ Plate frame: 1; ambient ▪ Snap-in, 8-well strips(white, sterile, cell-culture ready): 12; ambient