Constitutive Androstane Receptor-3 (CAR-3; NR1I3i3)
| Product Family | Product Number | Product Description | Technical Manual |
| IB0090 CAR-3 (NR1I3i3) |
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.
INDIGO’s cell-based reporter assays allow scientists to detect any biological activity that their test samples may exert against a specific receptor present in the cell. They utilize firefly luciferase reporter gene technology which provides superior precision and sensitivity. Since the receptor binding controls the expression of the luciferase reporter gene, luciferase activity in the cells can be correlated directly with the activity of the receptor. The strength of an interaction of a chemical with the target receptor is quantified using a luminometer to measure the level of light emitted.
Fast, reproducible, easy-to-analyze results are only four steps away
Many luciferase reporter assays require the user to grow their own cells and take time to optimize the results. INDIGO’s reporter cells contain the receptor of interest and the luciferase reporter gene. Reporter cells have been optimized to provide extreme sensitivity to quantify even small changes in receptor activity. With INDIGO’s cell-based reporter assays, the process is as easy as Thaw, Feed, Dose, and Read.
CAR3 Reporter Cells
CAR3 Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This proprietary cryopreservation process, enables long-term preservation of our unique reporter cells, so we can ship our cryopreserved reporter cells and assay reagents to you via overnight delivery, for your immediate use. Or, you can store the assay kits at -80°C. Once thawed, reporter cells are ready for immediate use so there is no need to take time on intermediate spin-and-wash steps, viability determinations, or cell titer adjustments.
CAR3 Luciferase Reporter
INDIGO’s CAR3 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.
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