Retinoic Acid Receptor Beta (RARβ; NR1B2)
|Product Family||Product Number||Product Description||Technical Manual|
|IB02101-32||Human RARβ Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB02101||Human RARβ Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB02102||Human RARβ Reporter Assay System, 1 x 384-well format assays||Technical Manual|
Retinoic Acid Receptor Beta Assay Kit
This Retinoic Acid Receptor Beta (RARβ) assay kit is an all-inclusive RAR beta reporter assay system that includes, in addition to RARβ 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.
RARβ 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 RAR beta 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.
RAR beta assay kits are offered in different assay formats to accommodate researchers’ needs: 3x 32, 1x 96, and 1x 384 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.
Retinoic Acid Receptor Beta 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.
Retinoic Acid Receptor Beta 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.
For more information on RARβ, visit the Nuclear Receptor Resource.
Synonyms: Retinoic Acid Receptor Beta, RARβ, RARb, RAR beta, Human Retinoic Acid Receptor Beta, Human RAR beta, NR1B2
Retinoic Acid Receptor Beta Assay Data
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.