Mouse/Rat PPARγ Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	MR00101	—
3 x-32 assays in-96 well format	MR00101-32	—

SIZE	SKU
1 x-96 well format assays	MR00101
3 x-32 assays in-96 well format	MR00101-32

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Specifications
Data
Target Background
Documentation
Overview
Citation
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Rodent (Mouse/Rat) Peroxisome Proliferator-Activated Receptor gamma (mrPPARg). INDIGO’s mrPPARg reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the mrPPARg. In addition to mrPPARg 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 mrPPARg. 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 Type		Nuclear Hormone Receptor, Nuclear Receptor Orthologs
Species		Rodent (Mouse / Rat shared ortholog)
Receptor Form		Hybrid
Assay Mode		Agonist, Antagonist
Kit Components		Rodent PPARg Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Rosiglitazone, (ref. agonist; in DMSO) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Validation of the mrPPARγ Assay was performed using the reference agonists Rosiglitazone (provided), Troglitazone (Tocris) and Ciglitazone (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). mrPPARγ Reporter Cells and Mock reporter cells were identically treated with Rosiglitazone, as described in Appendix 1. Luminescence was quantified using a GloMaxMulti+ plate-reading luminometer (Promega Corp.). Values of average Relative Light Units (RLU; average of n ≥ 6), respective standard deviation (SD), Signal-to-Background (S/B) and Coefficient of Variation (CV) were determined. 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.

Validation of Mouse/Rat PPARγ antagonist dose-responses performed in combination with INDIGO's Live Cell Multiplex Assay. Antagonist assays were performed using T0070907 (Tocris), and GW9662 (Tocris). 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).

Target Background

Peroxisome proliferator-activated receptor gamma (PPAR-gamma or PPARγ), also known as the glitazone receptor, or NR1C3 (nuclear receptor subfamily 1, group C, member 3) is a type II nuclear receptor that in humans is encoded by the PPARG gene. PPARs form heterodimers with retinoid X receptors (RXRs) and these heterodimers regulate transcription of various genes. PPARγ regulates adipocyte differentiation, fatty acid storage and glucose metabolism. The PPARγ knockout mice fail to generate adipose tissue when fed a high fat diet. Many insulin sensitizing drugs used in the treatment of diabetes target PPARγ as a means to lower serum glucose without increasing pancreatic insulin secretion. Additionally, PPARγ has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer. Alternatively spliced transcript variants that encode different isoforms have been described.

INDIGO’s Mouse/Rat PPAR gamma (mrpparγ; nr1c3) Assay utilizes proprietary mammalian cells engineered to provide high-level expression of mrPPARγ. Reporter Cells also incorporate an mrPPARγ-responsive luciferase reporter gene, therefore, quantifying expressed luciferase activity provides a sensitive surrogate measure of mrPPARγ activity in the treated cells. The principle application of this reporter assay system is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against mrPPARγ.