| Assay Method Information | |
| | Inhibitory Activity of Compounds Against hCYP11B2/hCYP11B1 |
| Description: | Seeding: Cells were cultured to a suitable state after resuscitation with maintenance medium, and 96-well flat plates were seeded with seeding medium according to 1×104/100 μL/well (unified cell volume).Changing solution: The supernatant was aspirated and discarded after seeding overnight (>12 hours) and adherence. 100-150 μL/well serum-free medium was added, and after being aspirated. After aspiration, 50 μL/well reaction medium was added for later use.Formulation: Compound was diluted with reaction medium containing 0.4 μM substrate (final experimental concentration is 0.2 μM):CYP11B2 substrate: 11-deoxycorticosterone (11-deoxycorticosterone, S4243, selleckchem). The final reaction concentration was 0.2 μMCYP11B1 substrate: 11-deoxycortisol (11-deoxycortisol, S4775, selleckchem). The final reaction concentration was 0.2 μM.Loading: The above compound dilutions were added to the cell plates at 50 μL/well. The background and control wells were set.Treating and sampling: The cell plates were incubated in a cell incubator for 16 hours after loading, and then each cell plate was centrifuged at 450 g for 2 min. 75 μL supernatant was transferred to a collection plate and frozen at −80° C. for future use (or direct detection).Detection: Homogeneous time-resolved fluorescence kit (Cisbio HTRF kit, Cat.64ALDPEG, Cat.62CRTPEG) was used to determine the concentration of aldosterone or cortisol in the supernatant. |
| Affinity data for this assay | |
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