Constitutive Androstane Receptor-3 (CAR-3; NR1I3i3)
|Product Family||Product Number||Product Description||Technical Manual|
|IB00901-32||Human CAR-3 Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB00901||Human CAR-3 Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB00302||Human CAR-3 Reporter Assay System, 1 x 384-well format assays|
Constitutive Androstane Receptor-3 Assay Kits
This Constitutive Androstane Receptor-3 (CAR-3) assay kit is an all-inclusive CAR-3 reporter assay system that includes, in addition to CAR-3 Reporter Cells, two optimized media for use during cell culture and (optionally) in diluting the test samples, a reference agonist, Luciferase Detection Reagent, a cell culture-ready assay plate, and a detailed protocol.
CAR3 Reporter Cells are prepared using INDIGO’s 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 pre- incubation of reporter cells 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 and 1x 96 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
CAR-3 assay kits also available in: Mouse
Constitutive Androstane Receptor-3 Assay Services
The primary application of INDIGO’s cell-based nuclear receptor assays are to quantitatively assess the bioactivity of a test compound as an agonist (activator) or antagonist (inhibition of an agonist response) of a given receptor. Service assays include a positive control reference compound and ‘vehicle’ control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online “Request a Quote” form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
CAR-3 Service Assays: Human, Mouse
Constitutive Androstane Receptor-3 Background
INDIGO’s Human Constitutive Androstane Receptor, isoform 3 (CAR3) Reporter Assay System utilizes proprietary mammalian cells engineered to provide high-level expression of human CAR3 (NR1I3 isoform 3), a ligand-activated transcription factor. The Reporter Cells utilize a modified version of human CAR3 in which the N-terminal DNA binding domain (DBD) has been replaced with the GAL4-DBD. The human CAR3 ligand binding domain (LBD) is unaltered and fully functional. 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 a direct interaction between a treatment compound and the nuclear receptor. Because this assay system expresses a GAL4-DBD + hCAR3 LBD hybrid receptor, the bio-activity of activators that act through indirect mechanisms (such as those that alter the phosphorylation status of the native N-terminal amino acid sequence of CAR3) may be dampened or go undetected.
Human CAR 3 is not constitutively active, rather, it exhibits ligand-dependent activation. The primary application of this reporter assay system is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert on human CAR3.
Unlike most nuclear receptors, this transcriptional regulator is constitutively active in the absence of ligand but is regulated by both agonists and inverse agonists. Ligand binding results in translocation of this protein to the nucleus, where it activates or represses target gene transcription. These ligands include bilirubin, a variety of foreign compounds, steroid hormones, and prescription drugs.
Constitutive Androstane Receptor-3 Assay Data
Agonist dose-response of the CAR3 Assay. Dose-response analysis of CAR3 Reporter Cells was performed using the reference agonist CITCO (provided). CITCO treatment media were prepared using serial 3-fold dilutions, as described in the Technical Manual. Luminescence was quantified and average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n = 3). Values of Fold-Activation and Z’ were calculated. The plot of ‘Fold-Activation vs. Log10[CITCO, nM]’ and EC50 determination were performed via least-squares non-liner regression using GraphPad Prism software.
Constitutive Androstane Receptor-3 Assay Research Areas
Xenobiotic Metabolism; Bile Acid and Xenobiotic; Toxicology; NAFLD/NASH; Drug-Nutrient Interaction