| Assay Method Information | |
| | Inhibition of Binding of E to ER |
| Description: | An ERE was cloned upstream of the luciferase gene, and MCF-7/ERE-luciferase monoclonal cells were selected by transfection of MCF-7 (TCHu74, National Collection of Authenticated Cell Cultures). The MCF-7/ERE-luciferase cells were seeded into a 96-well plate with an MEM (hyclone, SH30024.01B) medium containing 10% charcoal stripped FBS (Moregate, FBSF), 1% sodium pyruvate (sigma, S8636), 1% non-essential amino acids (sigma, M7145) and 500 μg/mL G418 at a density of 30,000 cells/well and cultured at 37° C. with 5% CO2. 20 mM stock solutions of the drugs are prepared, serially diluted 10-fold with 100% DMSO, and then diluted 20-fold with the medium. After the cells were cultured for 24 h, the medium was removed. 0.1 nM estradiol (sigma, E2758) and 10 μL of a medium-diluted drug were added to each well, and DMSO was added to the control group. The mixtures were well mixed by gentle shaking. The cells were cultured in an incubator at 37° C. with 5% CO2 for 24 h, and then the cell culture liquid was removed, followed by the addition of 50 μL of a luciferase substrate (Promega, E6110) to each well. The plate was let stand at room temperature in the dark for 10-15 min, and the chemiluminescence signal values were determined. |
| Affinity data for this assay | |
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