PPARd Rat Agonist Graph
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Rat PPARd 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 Rat Peroxisome Proliferator-Activated Receptor Delta. INDIGO’s rPPAR delta reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the rPPAR delta. In addition to rPPAR delta 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 rPPAR delta. 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
SpeciesRat
Receptor FormHybrid
Assay ModeAgonist, Antagonist
Kit Components
  • Rat PPARd Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • GW0742, (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

Dose-response analyses of rPPARδ Reporter Cells were performed using GW0742 (provided), GW501516 (Enzo) and L-165041 (Tocris). 10 mM master stocks were prepared in DMSO for each agonist. Final assay concentrations were prepared by serial dilution in 4-fold decrements, ranging between 10 µM and 0.15 nM. The highest assay concentration of residual DMSO was 0.1%, which has no effect on assay performance. To assess the level of background signal contributed by non-specific factors that cause gratuitous activation of the luciferase reporter gene, “mock” reporter cells were treated with GW0742 (mock reporter cells, which contain only the luciferase reporter vector, are not provided with assay kits). Treatment media were removed after 24 hr and LDR was applied. Luminescence was quantified using a GloMax-Multi+ luminometer (Promega). Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n ≥ 6). Fold-Activation (i.e., Signal-to-background; S/B) and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression and EC50 analyses were performed using GraphPad Prism software. RESULTS: The high S/B and Z’ values, low background and %CV values, and the absence of non-sp
Validation of rPPARδ Antagonist dose-responses performed in combination with INDIGO's Live Cell Multiplex Assay. (a.) Rat PPARδ antagonist assays performed using reference antagonists GSK0660, FH535, and GSK3787 (all from Tocris). (b.) To confirm that the observed drop in RLU values resulted from receptor inhibition, not induced cell death, the relative numbers of live cells in each assay well were determined at the end of the treatment period using INDIGO's Live Cell Multiplex (LCM) Assay (#LCM-01).

Target Background

INDIGO’s Rat PPAR delta (nr1c2; pparD/B; rPPARδ) Assay utilizes proprietary mammalian cells engineered to provide high-level expression of rPPARδ. Reporter Cells also incorporate a responsive luciferase reporter gene, therefore, quantifying expressed luciferase activity provides a sensitive surrogate measure of rPPARδ activity in the treated cells.

The principle 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 rat PPARδ.

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Peroxisome Proliferator-Activated Receptor Delta (PPARd, NR1C2)

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