Human PPARγ Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB00101	—
3 x-32 assays in-96 well format	IB00101-32	—
1 x-384 well format assays	IB00102	—

SIZE	SKU
1 x-96 well format assays	IB00101
3 x-32 assays in-96 well format	IB00101-32
1 x-384 well format assays	IB00102

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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 Human Peroxisome Proliferator-Activated Receptor Gamma. INDIGO’s Human PPAR gamma reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the Human PPAR gamma. In addition to PPAR gamma 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 human PPAR gamma.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

Ready to Use Upon Receipt
Includes All Needed Components
Contains Transfected Reporter Cells
Eliminates Cell Licensing Fees
Clear, Reproducible Results
Consistent Results Lot to Lot

Product Specifications

Target Type		Nuclear Hormone Receptor
Species		Human
Receptor Form		Hybrid
Assay Mode		Agonist, Antagonist
Kit Components		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
Orthologs Available		Yes
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Agonist dose-response analyses of the Human PPARγ Assay. Validation of the PPARγ Assay was performed using manual dispensing and following the protocol described in the assay Technical Manual, 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). PPARγ Reporter Cells and Mock reporter cells were identically treated with Rosiglitazone, as described in Appendix 1 of the technical manual. Luminescence was quantified using a GloMax-Multi+ 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. RESULTS: PPARγ reporter cells treated with 2,500 nM Rosiglitazone yielded an average RLU value with CV=7%, S/B = 162 and a corresponding Z’= 0.78. Similarly treated mock reporter cells demonstrate no significant background luminescence (≤ 0.05% that of ECMax). Thus, luminescence results strictly through ligand-activation of the PPARγ expressed in these reporter cells.

Antagonist dose-response analyses of Human PPARγ performed in combination with the INDIGO 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). Final assay concentrations of the respective antagonists ranged between 10 µM and 10 pM, including a 'no antagonist' control (n ≥ 6 per treatment; highest [DMSO] ≤ 0.15% f.c.). Each treatment also contained 220 nM (approximating EC50) Rosiglitazone as challenge agonist. Assay plates were incubated for 22 hrs, then processed according to the LCM Assay protocol to quantify relative numbers of live cells per treatment condition. Plates were then further processed to quantify PPARγ activity for each treatment condition. Averaged RFU values from each antagonist treatment group were normalized to the average RFU value of "no antagonist treatment" assay wells, which corresponds to 100% Live Cells in the LCM assay. Results: T0070907 and GW9662 both caused dose-dependent reduction in RLU values. The LCM Assay reveals no significant variance in the numbers of live cells per assay well, up to the maximum treatment concentration of 10 µM. Hence, the observed reduction in RLU values can be attributed to dose-dependent inhibition of PPARγ activity, and not to cell death. NOTE: RLU values will vary slightly between different production lots of reporter cells, and can vary significantly between different makes and models of luminometers.

Target Background

Peroxisome Proliferator-Activated Receptor Gamma (PPARγ), also known as the glitazone receptor, or NR1C3 is a type II nuclear receptor that in humans is encoded by the PPARγ 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 PPARγ Reporter Assay System utilize proprietary mammalian cells engineered to express human PPARG, commonly referred to as PPARγ.

The principle application of this assay product is in the screening of test samples to quantify functional activities, either agonist or antagonist, that they may exert against the human peroxisome proliferator-activated receptor gamma.