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Zebrafish GR Reporter Assay Kit

SIZE SKU PRICE
1 x-96 well format assays—
3 x-32 assays in-96 well format—
SIZE SKU
1 x-96 well format assays
3 x-32 assays in-96 well format
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Zebrafish Glucocorticoid Receptor. INDIGO’s zGR reporter assay utilizes proprietary non-human cells that have been engineered to provide constitutive expression of the zGR. In addition to zGR 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 zGR. 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
SpeciesZebrafish
Receptor FormNative
Assay ModeAgonist, Antagonist
Kit Components
  • Zebrafish GR Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • Dexamethasone, (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

Activity dose-response of zfGR using various reference agonists. All dose-response analyses of zfGR were performed according to the protocol provided in this Technical Manual. (A.) Agonist Dose-responses: Reporter cells were treated with the reference agonists Dexamethasone (provided), Beclomethasone dipropionate (Sigma), Mometasone furoate, Fluticasone propionate, Budesonide, Methylprednisolone, GSK 9027, and Hydrocortisone (all from Tocris). Luminescence was quantified at the 24 hr assay endpoint and values of average Fold-Activation and Z’ were calculated as described by Zhang, et al. (1999)1. Data are plotted as Fold-Activation vs. Log10[Agonist, pM]. The EC50 for dexamethasone is ~ 150 pM, with the 500 pM treatment yielding ≥ 800-fold activation over the untreated reporter cells, and the corresponding Z’= 0.92. (B.) Antagonist Dose-responses: Reporter cells were co-treated with a fixed EC80 concentration of the agonist Dexamethasone and varying concentrations the GR antagonists Mifepristone(Tocris) and Cyproterone Acetate (Santa Cruz). Data are plotted as RLU vs. Log10[Antagonist, nM]. The IC50 for Mifepristone is ~ 0.5 nM; Z’ = 0.84 at 150 nM. Non-linear regression and EC50 analyses were performed using GraphPad Prism software.

Target Background

INDIGO’s Zebrafish Glucocorticoid Receptor (nr3c1) nuclear receptor assay utilizes proprietary mammalian cells engineered to provide constitutive high level expression of full-length, unmodified Zebrafish (Danio rerio) Glucocorticoid Receptor (nr3c1), a ligand-dependent transcription factor, commonly referred to as GR.

INDIGO’s Reporter Cells include the luciferase reporter gene functionally linked to a GR-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in zfGR activity. Luciferase gene expression occurs after ligand-bound zfGR undergoes DNA binding, recruitment and assembly of the co-activators and accessory factors required to form a functional transcription complex, culminating in expression of the reporter gene. Unlike some other cell-based assay strategies, the readout from INDIGO’s reporter cells demands the same orchestration of all intracellular molecular interactions and events that can be expected to occur in vivo

Also available as a service

Glucocorticoid Receptor (GR, NR3C1)

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