| Assay Method Information | |
| | Human Glucocorticoid Receptor (hGR) Ligand-Binding Assay |
| Description: | The human lymphoblast cell line IM9 (ATCC, Bethesda, MD) was cultivated in RPMI 1640 media containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 2 mM L-glutamine at 37° and 7% CO2 in a humidified incubator. Cells were centrifuged for 10 minutes at 1500 g and were washed in PBS and repelleted. Cell were then resuspended in homogenization buffer consisting of: 10 mM TES, 10 mM sodium molybdate, 1 mM EDTA, pH 7.4, 20 mM 2-mercaptoethanol, and 10% glycerol. Disruption of the cells was performed by nitrogen cavitation using 2×15 minutes at 600 to 750 psi nitrogen in a N2 cavitator at 0° C. The cell preparation was then centrifuged at 27,000 g for 15 minutes, and the resultant supernatant (=cytosol of IM9 cells) was centrifuged at 103,000 g for 60 minutes at 4° C. The amount of protein in the supernatant fraction was determined using a BCA assay kit and aliquots were snap frozen in a dry ice-acetone bath and stored at −70° C. Competitive binding assays were done in duplicate in homogenization buffer with a total volume of 200 μl. To this end, 1 mg of IM9 cytosol, 0.05 μCi (1.5 nM) of 3H-dexamethasone and unlabeled Example compounds as competitor compounds at 1 μM were mixed. The reaction was stopped after incubation at 0° C. for 16 to 18 hours by the addition of 100 μl of a charcoal-dextran mixture (2% activated charcoal, 0.5% dextran in 10 mM Tris, 1 mM EDTA, pH 7.4). Another incubation step at 0° C. for 10 minutes followed before the samples were centrifuged for 5 minutes at 8200 g. 100 μl of the supernatant) was finally assayed for radioactivity by liquid scintillationspectrometry, and percentage inhibition of 3H-dexamethasone binding was calculated. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |