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Mouse CAR Reporter Assay Kit

SIZES SKU
1 x 96-well format assays M00901
3 x 32 assays in 96-well format M00901-32
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Mouse Constitutive Androstane Receptor (mCAR). INDIGO’s mCAR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the mCAR. 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, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against mCAR. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeNuclear Hormone Receptor, Nuclear Receptor Orthologs
SpeciesMouse
Receptor FormHybrid
Assay ModeAgonist, Antagonist
Kit Components
  • Mouse CAR Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • TCPOBOP (ref. agonist; in DMSO)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Shipping RequirementsDry Ice
Storage temperature-80C

Data

TCPOBOP agonist dose-response of the mCAR Assay. Dose-response analyses of mCAR Reporter Cells were performed according to the protocol provided in the 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 uM 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.

Target 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.

Citations

Tris(1,3-dichloro-2-propyl) phosphate is a metabolism-disrupting chemical in male mice

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant. The primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), is detectable in the urine of over 90 % of Americans. Epidemiological studies show sex-specific associations between urinary BDCPP levels and metabolic syndrome, which is an established risk factor for type 2 diabetes, heart disease, and stroke. We used a mouse model to determine whether TDCPP exposure disrupts glucose homeostasis. Six-week old male and female C57BL/6J mice were given ad libitum access to diets containing vehicle (0.1 % DMSO) and TDCPP resulting in the following treatment groups: 0 mg/kg/day, 0.02 mg/kg/day, 1 mg/kg/day, or 100 mg/kg/day. After being on the experimental diet for five weeks without interruption, body composition was analyzed, glucose and insulin tolerance tests were performed, and fasting glucose and insulin levels were quantified. TDCPP at 100 mg/kg/day caused male sex-specific adiposity, fasting hyperglycemia, and insulin resistance. TDCPP-induced modulation of nuclear receptor activation was investigated using an in vitro screen to identify potential mechanisms of metabolic disruption. TDCPP activated farnesoid X receptor (FXR) and pregnane X receptor (PXR), and inhibited the androgen receptor (AR). PXR target genes, but not FXR target genes, were upregulated in livers from mice exposed to 100 mg TDCPP/kg/day. Interestingly, PXR target genes were differentially expressed in livers from both males and females. It remains to be determined whether TDCPP-induced metabolic disruption occurs via modulation of nuclear receptor activity. Taken together, these studies build upon the association of TDCPP exposure and metabolic syndrome in humans by identifying sex-specific effects of TDCPP on glucose homeostasis in mice.
2022-12-07

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Constitutive Androstane Receptor (CAR, NR1I1)

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