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Human RARβ Reporter Assay Kit

1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays
1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Retinoic Acid Receptor Beta (RARb). INDIGO’s RAR Beta reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the RAR Beta. In addition to RAR Beta 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 human RAR Beta. 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.


  • Ready to Use Upon Receipt

  • Includes All Needed Components
  • Contains Transfected Reporter Cells
  • Eliminates Cell Licensing Fees
  • Clear, Reproducible Results
  • Consistent Results Lot to Lot

Product Specifications

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


Agonist dose-response analyses of the RARβ Assay. Validation of the RARβ Assay was performed using manual dispensing and following the protocol described in the assay Technical Manual, using the reference agonists all-trans-Retinoic Acid (provided), Adapalene (Tocris), and BMS 453 (Tocris). In addition, to assess the level of background signal contributed by non-specific factor(s) that may cause activation of the luciferase reporter gene, “mock” reporter cells were specially prepared to contain only the luciferase reporter vector (mock reporter cells are not provided with assay kits). RARβ Reporter Cells and Mock reporter cells were identically treated with trans-retinoic acid, as described in Appendix 1 of the technical manual. Luminescence was quantified using a GloMax-Multi+ plate-reading luminometer (Promega Corp.). Average relative light units (RLU) and respective standard deviation (SD) and Signal-to-Background (S/B) values were determined for each treatment concentration (n ≥ 6). Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression analyses were performed and EC50 values determined using GraphPad Prism software. Mock reporter cells treated with trans- retinoic acid demonstrate no significant background luminescence (≤ 0.05% that of the reporter cells at ECMax). Thus, luminescence results strictly through ligand-activation of the human RARβ expressed in these reporter cells. These data confirm the robust performance of this RARβ Reporter Assay, and demonstrate its suitability for use in HTS applications.
Validation of RARβ antagonist dose-responses performed in combination with INDIGO's Live Cell Multiplex Assay. RARβ antagonist assays were performed using LE 135 (Tocris), and CD 2665 (Tocris). Assay setup and quantification of RARβ activity were performed following the protocol described in this Technical Manual. To confirm that the observed drop in RLU values resulted from receptor inhibition, as opposed to induced cell death, the relative numbers of live cells in each assay well were determined using INDIGO's Live Cell Multiplex (LCM) Assay (#LCM-01). Final assay concentrations of the respective antagonists ranged between 6 µM and 5.7 pM, including a 'no antagonist' control (n ≥ 6 per treatment; highest [DMSO] ≤ 0.15% f.c.). Each treatment also contained 3 nM (approximating EC50) trans-Retinoic Acid as challenge agonist. Assay plates were incubated for 24 hrs, then processed according to the LCM Assay protocol to quantify relative numbers of live cells per treatment condition. Plates were then further processed to quantify RARβ activity for each treatment condition. Results: LE 135 and CD 2665 both caused dose-dependent reduction in RLU values. The LCM Assay reveals no significant variance in the numbers of live cells per assay well, up to the maximum treatment concentration of 6 µM. Hence, the observed reduction in RLU values can be attributed to dose-dependent inhibition of RARβ activity, and not to cell death.

Target Background

Retinoic acid receptors (RARs) are nuclear hormone receptors of the NRB1 class, which function as heterodimers with retinoid X receptors (RXRs). There are three distinct RAR subtypes; RARalpha, RARbeta and RARgamma. RARalpha is present in most tissue types, whereas RARbeta and RARgamma expression is more selective. RXR-RAR heterodimers act as ligand-dependent transcriptional regulators by binding to the specific retinoic acid response element (RARE) found in the promoter regions of target genes. In the absence of an RAR agonist, RXR-RAR recruits co-repressor proteins such as NCoR and associated factors such as histone deacetylase to maintain a condensed chromatin structure. RAR agonist binding stimulates co-repressor release and co-activator complexes, such as histone acetyltransferase, are recruited to activate transcription. RARs transduce retinoid signals in vivo, which mediates proper embryogenesis, differentiation and growth arrest. Specifically, RXRalpha-RARgamma heterodimers are necessary for growth arrest and viseral and primitive endodermal differentiation, whereas RXRalpha-RARalpha is required for cAMP-dependent parietal endodermal differentiation. In vitro it has been difficult to elucidate the roles of individual subtypes as functional RAR knockouts generate artificial redundancies that are thought not to exist under normal conditions.

INDIGO’s Human Retinoic Acid Receptor Beta (RARβ) Reporter Assay System utilizes proprietary mammalian cells engineered to provide constitutive, high-level expression of human RARβ (NR1B2), a ligand-dependent transcription factor. Because these cells incorporate a responsive luciferase reporter gene, quantifying expressed luciferase activity provides a sensitive surrogate measure of RARβ activity in treated cells.

The primary application of this reporter assay system is in the screening of test samples to quantify functional activity, either agonist or antagonist, that they may exert against human RARβ.


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.
All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

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Retinoic Acid Receptor Beta (RARb, NR1B2)

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