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| | Cellular Inhibition Assay |
| Description: | V79MZ cells expressing human CYP11B1 and human CYP11B2 genes, respectively, were grown on 24-well cell culture plates (8×10^5 cells per well) with 1.9 cm^2 culture area per well in 1 ml DMEM culture medium until confluence. Before testing, the DMEM culture medium was removed and 450 μl of fresh DMEM, containing the inhibitor in at least three different concentrations for determining the IC50 value, was added to each well. Every value was determined at least three times. After a pre-incubation step of 60 min at 37° C., the reaction was started by the addition of 50 μl of DMEM containing the substrate 11-deoxycorticosterone (containing 0.15 μCi of [1,2-3H] 11-deoxycorticosterone, dissolved in ethanol, final concentration 100 nM). The V79MZh11B1 cells were incubated for 25 min, the V79MZh11B2 cells were incubated for 50 min. Controls were treated in the same way without inhibitors. The maximum DMSO concentration in each well was 1%. Enzyme reactions were stopped by extracting the supernatant with 500 μl ethyl acetate. Samples were centrifuged (10000×g, 2 min), and the solvent was pipetted into fresh cups. The solvent was evaporated and the steroids were redissolved in 40 μl of methanol and analyzed by HPLC using radioflow detection (Ehmer et al. J. Steroid Biochem. Mol. Biol. 2002, 81, 173-179 and Denner et al. Endocr. Res. 1995, 21, 443-448). |
| Affinity data for this assay | |
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