| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | GnRH/HEK293 (human embryonic kidney cell 293) cultured cells in the logarithmic growth phase were washed with DPBS (Dulbecco's phosphate buffered saline) buffer, and an appropriate amount of 0.05% EDTA (ethylenediaminetetraacetic acid)-trypsin was added. The cells were placed in a carbon dioxide incubator at 37° C. for digestion for 1-2 minutes, and then removed from the incubator. A medium was added to the cells to terminate the digestion. Cells were dispersed by repeated pipetting and harvested by centrifugation. The cells were seeded into a 384-well Poly-Lysine-Coated. Cell Plate at a density of 20,000 cells per well. 20 μL, and incubated overnight in a 5% CO2, 37° C. incubator.[0177]On the next day, 20 μL, of 2×Fluo-4Direct™ buffer was added to each well, and the plate was incubated in a 5% CO2, 37° C. incubator for 50 minutes. The cells were placed at room temperature for 10 minutes. 0.2 nM Leuprolide acetate was diluted to 10 concentrations by 4-fold serial dilution in ECHO, and 900 nL of that was transferred to the compound plate. 30 μL of FLIPR buffered saline was added to the compound plate, and the plate was centrifuged at 1000 rpm for 1 min. The software of the FLIPR instrument was run, and 10 μL of assay buffer salt solution was added according to the set program. The fluorescence signal was read. Then 10 μL of the reference compound as an agonist was added, and the fluorescent signal was read. EC50 was calculated, and the agonist with a concentration of 6×EC50 was prepared.2 mM assay compounds and an appropriate concentration of reference compound were serially diluted 4-fold to 10 concentrations in ECHO, and 900 nL of that was transferred to a compound plate. 30 μL of FLIPR buffered saline was added to the compound plate, and the plate was centrifuged at 1000 rpm for 1 min. The software of the HIM instalment was run, and 10 μL of assay and reference compounds were added to the cell plate according to the set program. The fluorescent signal was read. Then 10 μL of agonist with a concentration of 6×EC50 was added to the cell plate and the fluorescent signal was read.IC50 of the compound to inhibit the calcium flow of gonadotropin-releasing hormone receptor, i.e., the drug concentration when the Ca2+ flow was inhibited by half in the cells stably expressing the GnRH receptor, was calculated. The IC50 of the drug was calculated by GraphPad Prism 5.0 software. |
| Affinity data for this assay | |
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