Zebrafish Aryl Hydrocarbon Receptor (zAhR)
|Product Family||Product Number||Product Description||Technical Manual|
|Z06001-32||Zebrafish AhR Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|Z06001||Zebrafish AhR Reporter Assay System, 1 x 96-well format assays||Technical Manual|
Zebrafish Aryl Hydrocarbon Receptor Assay Kit
This Zebrafish Aryl Hydrocarbon Receptor (zAhR) assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition to zAhR reporter cells, two optimized media for use during cell culture and in diluting the user's test samples, the AhR reference agonist MeBIO, Luciferase Detection Reagent, and a cell culture-ready assay plate.
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.
Zebrafish 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.
Zebrafish Aryl Hydrocarbon Receptor Assay Background
While technically not a nuclear receptor, the AhR is mechanistically and functionally similar to members of that super-family, being both a receptor and a ligand-activated transcription factor. More formally, the AhR is a member of the basic helix-loop-helix, Per-Arnt-Sim family of transcription factors. As its name implies, the Aryl Hydrocarbon Receptor (AhR) is a xenobiotic-sensing receptor. It is responsible for the adverse toxicologic effects elicited by various polycyclic aromatic hydrocarbon environmental and industrial pollutants, perhaps the most infamous being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
The AhR is present in the cytosol where, in the non-active state, it is in a complex with chaperone proteins such as Hsp90. Binding of a polycyclic aromatic hydrocarbon to AhR leads to nuclear translocation and association with its partner protein ARNT. The AhRARNT hetero-dimer binds to specific cognate DNA sequence elements known as dioxin/xenobiotic response elements (DRE/XRE) present in the regulatory region of a variety of target genes. Binding of AhR:ARNT to these elements, and subsequent recruitment of transcription co-activator complexes, induces the transcription of a battery of target genes, including several cytochrome P450 genes. Other target genes of the TCDD/AhR-complex encode inhibitory or stimulatory growth factors that mediate cellular growth and differentiation.
INDIGO's Zebrafish Aryl Hydrocarbon Receptor (zfAhR) Reporter Cells are constructed in proprietary zebrafish cells. This zebrafish cell line has been modified to contain the luciferase reporter gene functionally linked to tandem DRE/XRE genetic response elements and a minimal promoter sequence. 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 the native zebrafish AhR.
For more information on AhR, visit the Nuclear Receptor Resource.
Agonist dose-response analyses of Zebrafish AhR. Agonist analyses of zfAhR Reporter Cells were performed according to the protocol described in the assay technical manual, using the reference agonists MeBIO (provided), TCDD (2,3,7,8-Tetrachlorodibenzo-P-dioxin (6-Formylindolo(3,2-b)carbazole; Cambridge Isotope Laboratories), ITE (2-(1H-indole-3-ylcarbonyl)-4-thiazolecarboxylic methyl ester; Tocris), and Indirubin (Cayman). Luminescence was quantified and average relative light units (RLU), Fold-Activation, and corresponding values of standard deviation (SD) and coefficient of variation (%CV) were determined for each treatment concentration (n ≥ 3). Z’ values were calculated as described by Zhang, et al. (1999).
Antagonist dose-response analyses of Zebrafish AhR. Antagonist analyses of zfAhR Reporter Cells were performed according to the protocol described in the assay technical manual, using the reference antagonists GNF351 (Calbiochem). Non-linear regression and EC50 analyses were performed using GraphPad Prism software.
Zebrafish Aryl Hydrocarbon Receptor Assay Research Areas
Xenobiotic Metabolism; Bile Acid and Xenobiotic; Toxicology; Drug-Nutrient Interaction; Environmental Toxicology