| Assay Method Information | |
| | Inhibition of Activity of 5-HT7 Serotonin Receptor |
| Description: | For luminescence-based cAMP assay, HEK293 cells were cultured on a 150-mm dish. Prior to transfection, the culture medium was replaced from DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin to DMEM containing 10% dialyzed FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. 4 hours later, transfection was conducted using 10 μg of a human 5-HT7R plasmid and 10 μg of a GloSensor-22F plasmid (Promega). The transfected cells were prepared on a white, flat, clear-bottom, 384-well plate (Greiner) (15,000 cells/well, 20 μL/well). 6 hours later, after removing the culture medium, the cells were treated with 20 μL of a 3% Glosensor cAMP reagent containing luciferin in a 1×HBSS, 1 M HEPES, pH 7.4 buffer. In addition, the compound of each of the examples was prepared at different concentrations in an assay buffer containing 0.1% bovine serum albumin. 30 minutes later, 10 μL of the compound solution was treated to the cells. After measuring luminescence using a Tecan microplate reader (Spark), IC50 value was obtained using the Prism 8.0 program (GraphPad Software). |
| Affinity data for this assay | |
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