Mouse Aryl Hydrocarbon Receptor (mAhR)
|Product Family||Product Number||Product Description||Technical Manual|
|M06001-32||Mouse AhR Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|M06001||Mouse AhR Reporter Assay System, 1 x 96-well format assays||Technical Manual|
Mouse Aryl Hydrocarbon Receptor Assay Kit
This Mouse Aryl Hydrocarbon Receptor (mAhR) assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition to mAhR Reporter Cells, two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist (MeBIO), Luciferase Detection Reagent, and a cell culture-ready assay plate.
mAhR Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields exceptional cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, cell titer adjustments, or the pre-incubation of reporter cells prior to assay setup.
INDIGO’s mAhR 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.
mAhR assay 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.
Mouse Aryl Hydrocarbon 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.
Mouse Aryl Hydrocarbon Receptor Background
The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic helix loop helix- PER-ARNT-SIM (bHLH-PAS) family of transcription factors. It was initially characterized by its ability to sense xenobiotics, particularly environmental contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, also known as dioxin) and polycyclic hydrocarbons (PAHs), such as benzo[a]pyrene. Recent interest in AhR has also focused on its diverse physiological functions in a variety of systems, including but not limited to the liver, intestine, skin, and immune system.
The prototypical signaling pathway of AhR is similar to that of the nuclear receptor superfamily. In the absence of ligand, AhR is located in the cytoplasm, bound to a chaperone complex including a dimer of Hsp90, AIP, and p23. When bound to a ligand, the AhR translocates into the nucleus, where it binds to its heterodimerization partner, ARNT. The AhR-ARNT complex then binds specific cognate DNA sequence elements known as dioxin/xenobiotic/AHR response elements (DRC/XRE/AHRE), located upstream of responsive genes, most notable of which are members of the cytochrome P450 family of phase I drug metabolizing enzymes (predominantly CYP1A1, CYP1A2, and CYP1B1).
Differences in relative affinity for TCDD between species are known to exist. For example, the mouse AhRb-1 allele binds TCDD with 10-fold higher relative affinity than human AhR.3 For this reason, mouse AhR can be used as a sensitive tool for detecting the presence of dioxins and similar environmental pollutants.
INDIGO's Mouse Aryl Hydrocarbon Receptor (AhR) Reporter Cells include the luciferase reporter gene functionally linked to an AhR-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in mouse AhR activity.
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 mouse AhR.
For more information on AhR, visit the Nuclear Receptor Resource.
Synonyms: Aryl Hydrocarbon Receptor, AhR, Ah Receptor, Dioxin Receptor
Mouse Aryl Hydrocarbon Receptor Assay Data
Agonist dose-response analyses of Mouse AhR. Agonist analyses of Mouse AhR Reporter Cells were performed using the reference agonist MeBIO (provided) and a.) examples of environmental contaminants of concern: TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin; Cambridge Isotope Laboratories, Inc.), Benzo[a]pyrene (Sigma-Aldrich), and PCB126, or b.) prototypical activators of AhR that are of nutritional or pharmaceutical relevance: FICZ (6-Formylindolo[3,2-b]carbazole; Cayman Chemical), ITE (2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic methyl ester; Cayman Chemical), β-Naphthoflavone (Sigma-Aldrich), and Indirubin. Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n = 3 / conc.). Values of Fold-activation and Z’ were calculated as described by Zhang, et al. (1999). Least squares fit non-linear regression and EC50 analyses were performed using GraphPad Prism software. The reference agonist MeBIO yielded an EC50 ~ 34 nM, and a Z' value of 0.85, confirming the robust performance of this assay, and its suitability for HTS.
Mouse AhR Antagonist dose-response analyses. Antagonist analyses of Mouse AhR Reporter Cells were performed according to the protocol described in this Technical Manual. Briefly, Mouse AhR Reporter Cells were co-treated with an EC80 concentration of the reference agonist MeBIO (provided) and varying concentrations of the AhR antagonists GNF351 (Calbiochem), CH223191 (Tocris), and 3’,4’-Dimethoxyflavone (Sigma). INDIGO’s Live Cell Multiplex (LCM) Assay confirmed that no treatment concentrations were cytotoxic (data not shown). Non-linear regression analyses of RLU vs. Log10[Antagonists, nM] were plotted and IC50 determination made for GNF351 using GraphPad Prism software.