Peroxisome Proliferator-Activated Receptor Gamma (PPARγ; NR1C3)
|Product Family||Product Number||Product Description||Technical Manual|
|IB00101-32||Human PPARγ Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB00101||Human PPARγ Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB00102||Human PPARγ Reporter Assay System, 1 x 384-well format assays||Technical Manual|
Peroxisome Proliferator-Activated Receptor Gamma Assay Kit
This Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) assay kit is an all-inclusive PPAR gamma reporter assay system that includes, in addition to PPARγ Reporter Cells, two optimized media for use during cell culture and (optionally) in diluting the test samples, a reference agonist, Luciferase Detection Reagent, a cell culture-ready assay plate, and a detailed protocol.
INDIGO’s cell-based reporter assays allow scientists to detect any biological activity that their test samples may exert against a specific receptor present in the cell. They utilize firefly luciferase reporter gene technology which provides superior precision and sensitivity. Since the receptor binding controls the expression of the luciferase reporter gene, luciferase activity in the cells can be correlated directly with the activity of the receptor. The strength of an interaction of a chemical with the target receptor is quantified using a luminometer to measure the level of light emitted.
Fast, reproducible, easy-to-analyze results are only four steps away
Many luciferase reporter assays require the user to grow their own cells and take time to optimize the results. INDIGO’s reporter cells contain the receptor of interest and the luciferase reporter gene. Reporter cells have been optimized to provide extreme sensitivity to quantify even small changes in receptor activity. With INDIGO’s cell-based reporter assays, the process is as easy as Thaw, Feed, Dose, and Read.
PPARγ Reporter Cells
PPARγ Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This proprietary cryopreservation process, enables long-term preservation of our unique reporter cells, so we can ship our cryopreserved reporter cells and assay reagents to you via overnight delivery, for your immediate use. Or, you can store the assay kits at -80°C. Once thawed, reporter cells are ready for immediate use so there is no need to take time on intermediate spin-and-wash steps, viability determinations, or cell titer adjustments.
PPARγ Luciferase Reporter
INDIGO’s PPARγ 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.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
PPARγ assay kit also available in: Mouse/Rat Shared Ortholog, Cyn Monkey, Zebrafish
Peroxisome Proliferator-Activated Receptor Gamma 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.
PPARγ Service Assays: Human, Rat/Mouse Shared Ortholog, Cyn Monkey, Zebrafish
Peroxisome Proliferator-Activated Receptor Gamma Assay 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.
Synonyms: Peroxisome Proliferator-Activated Receptor Gamma, PPARγ, PPARg, PPAR gamma, Human Peroxisome Proliferator-Activated Receptor Gamma, hPPARγ, NR1C3
Peroxisome Proliferator-Activated Receptor Gamma Assay 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.