Puracyp`s nuclear receptor activation kits are all-inclusive cell-based assay systems. The kits incorporate mycoplasm-free, stably-transfected cells derived from hepatoma cell lines that have been treated with a cell-arrest agent to prevent further proliferation. Exposure to the cell-arrest agent ensures that the cells in the kits can only be used for a single 96-well assay. The kits also include a cell culture-ready assay plate, optimized cell culture medium, cell dosing medium, a positive control specific for the nuclear receptor, CellTiter-Fluor for determining cell viability, luciferase detection reagents and, if so ordered, a P450-Glo™ substrate for assessing induction of CYP450 enzyme activity. The primary application of these assay kits is to identify agents with the ability to activate PXR and/or AhR, thereby inducing drug-metabolizing enzymes and ABC transporters.
Puracyp`s Human AhR Activation Assay System utilizes 1A2-DRE™ cells constructed with a proprietary process for producing stably-transfected cell lines. 1A2-DRE™ cells harbor the human AhR gene and a luciferase reporter gene linked to the human CYP1A2 promoter and 3 copies of the dioxin response element (DRE) enhancer. The 1A2-DRE™ cells included in the assay kit remain highly viable upon thawing, and can be immediately dispensed into 96-well plates. There is no need for intermediate spin-and-wash steps. However, determining viability and cell titer is recommended. A list of references describing the use of Puracyp`s 1A2-DRE7U cells and other stable cell lines is given in this manual.
Puracyp`s Human AhR Activation Assay System also includes CellTiter-Fluor™, a cell viability detection reagent, and ONE-Glo™, a luciferase detection reagent, from Promega Corporation. Since the luciferase reporter gene is stably integrated into Puracyp`s 1A2-DRE"* cells, the assay gives extremely low background, high sensitivity, and a broad linear dynamic range typical of bio-luminescence reporter gene technology. Further details on the use of CellTiter-Fluor™, ONE-GloT* and the P450-Glo™ CYP1A2 Assay with Luciferin-1A2 can be found at www.promega.com.
In a typical assay, 1A2-DRE™ cells are first dispensed into the wells of the assay plate, and then placed in a CO2 incubator at 37°C to equilibrate overnight. The following day, the cells are treated with user`s test compounds and the appropriate positive control. Since the 1A2-DRE™ cells included with the kit are non-proliferative, test treatment should take place within 24 h of plate seeding. Once treated, the cells are returned to the CO2 incubator at 37 C for 24 h. The dosing medium is then discarded, cell viability is assessed fluorimetrically with CellTiter-Fluor7*, and luciferase activity then measured using ONE- Glo™. The luminescence light intensity is directly proportional to the extent of AhR activation and accompanying gene transcription in the 1A2-DRE™ cells.