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 384--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 384--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--DRE™ 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--Glo™ and the P450--Glo™ CYP1A2 Assay with Luciferin--IPA 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 37oC 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 37oC for 48 h. The dosing medium is then discarded, cell viability is assessed fluorimetrically with CellTiter--Fluor™, 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.