| Assay Method Information | |
| | hPXR Tranxactivation Assays |
| Description: | The hPXR transactivation assays (PXR agonistic and antagonistic assays) were performed in the HepG2 cells stably expressing FLAG-hPXR and CYP3A4-luciferase reporter. Briefly, various concentrations (1-to-3 series dilutions for 10 concentration levels from 40 μM with 0.5% final DMSO concentration) of hPXR agonist rifampicin, various concentrations of tested chemicals alone or combined with 5 μM of rifampicin, were added to the wells of white 384-well tissue culture-treated plates with 5,000 cells in 25 μl of phenol red-free DMEM supplemented with 5% charcoal/dextran-treated FBS and incubated for 24 h at 37° C. before luciferase assay using Steadylite HTS (PerkinElmer Life Sciences). The luminescence signal was detected using an Envision plate reader (PerkinElmer Life Sciences). In the agonistic assays, DMSO (0.5% final concentration) was used as the negative control (0% activation) and rifampicin (10 μM in 0.5% DMSO) was used as the positive control (100% activation). In the antagonistic assays, rifampicin (5 μM with 0.5% final DMSO concentration) was used as the negative control (0% inhibition) and DMSO (0.5%) was used as the positive control (100% inhibition). The activities of individual chemicals tested at various concentrations were normalized to the corresponding positive and negative controls to generate % Activation in agonistic assays and % Inhibition in antagonistic assays. The curve-fitting software GraphPad Prism 4.0 (Graphpad Software, La Jolla, Calif.) was used to generate the dose response curves and determine the IC50 (for hPXR antagonists) or EC50 (for hPXR agonists) values for individually tested compounds if applicable. |
| Affinity data for this assay | |
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