| Assay Method Information | |
| | HSD2 Activity |
| Description: | Human descending colon epithelial stem cells were cultured as 3D organoids in accordance with Sato et al Gastroenterology. 2011 November; 141(5): 1762-72. Organoids were dissociated using TrypLE Express (life technologies) and plated on 96-well transwells (corning) in supplemented basal media (SBM-advanced DMEM/F12 containing 10 mM HEPES, 1:100 Glutamax, 1:100 penicillin/streptomycin, 1:100N2, 1:50 B27, 1 mM N-acetylcysteine, 10 nM [Leu15]-gastrin I) containing 100 ng/mL Wnt3A (W), 50 ng/mL EGF (E), 100 ng/mL Noggin (N), 500 ng/mL RSpondinl (R), 500 nM A83-01 (S) and 2.5 uM thiazovivin (T). Cultures were differentiated using SBM containing ENRA and 30 nM aldosterone on day 3, and cultures were used for assay on day 6 or 7. Compounds were diluted in DMSO and serial dilutions prepared by titrating in DMSO. Compounds were then diluted into DMEM/F12. Transwell plates containing descending colon cultures were washed twice with DMEM/F12 and compound was added to the apical compartment. Cells were incubated with test compound for 30 minutes at 37 C., 5% CO2 to equilibrate across the cell membrane. A second compound plate was prepared in which the serially diluted compounds in DMSO were diluted into DMEM/F12 containing 40 nM cortisol. Following the 30 minute pre-incubation step, the apical media was aspirated and compounds diluted in DMEM/F12 with 40 nM cortisol were added to the apical side of the transwell. The plate was then incubated for four hours at 37 C., 5% CO2. Cortisol levels were measured using a cortisol HTRF assay kit as described by the manufacturer (Cisbio). Concentration-response curves were then plotted and IC50 (and pIC50) values were determined using least squares non-linear regression. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |