Mouse Constitutive Androstane Receptor (mCAR; nr1i3)
|Product Family||Product Number||Product Description||Technical Manual|
|M00901-32||Mouse CAR Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|M00901||Mouse CAR Reporter Assay System, 1 x 96-well format assays||Technical Manual|
Mouse Constitutive Androstane Receptor Assay Kit
This Mouse Constitutive Androstane Receptor (mCAR) assay kit is an all-inclusive firefly luciferase reporter assay system that includes, in addition to mCAR Reporter Cells, two optimized media for use during cell culture and (optionally) in diluting the test samples, a reference agonist (TCPOBOP), Luciferase Detection Reagent, a cell culture-ready assay plate, and a detailed protocol.
mCAR Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation 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.
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 assay kits also available in: Human
Mouse Constitutive Androstane Receptor 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.
mCAR Service Assays: Human, Mouse
Mouse Constitutive Androstane Receptor Background
INDIGO’s Mouse Constitutive Androstane Receptor (nr1i3) Assay is a cell-based genetic reporter assay that utilizes proprietary 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.
For more information on CAR, visit the Nuclear Receptor Resource.
Mouse Constitutive Androstane Receptor Assay Data
TCPOBOP agonist dose-response of the mCAR Assay. Dose-response analyses of mCAR Reporter Cells were performed according to the protocol provided in this Technical Manual. mCAR Reporter Cells were treated with the reference agonists TCPOBOP (provided) and Meclizine (Tocris). Luminescence was quantified using a GloMax-Multi+ luminometer. Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n ≥ 6), and Fold-Activation (i.e., signal-to-background) and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression analyses and EC50 determination were performed using GraphPad Prism software. RESULTS: mCAR reporter cells treated with 6.0 µM TCPOBOP yielded a S/B of ~ 6 and a corresponding Z’= 0.82. These data confirm the robust performance of this mCAR assay, and demonstrate its suitability for use in HTS applications. NOTE: RLU values will vary slightly between different production lots of reporter cells, and can vary significantly between different makes and models of luminometers.
Mouse Constitutive Androstane Receptor Assay Research Areas
Xenobiotic Metabolism; Bile Acid and Xenobiotic Metabolism; Toxicology; NASH/NAFLD; Drug-Nutrient Interaction