| Assay Method Information | |
| | In Vitro Binding Assays Radioligand Binding Assay |
| Description: | Radioligand binding assays were performed using [3H]5-HT as the radioligand using cell membranes prepared from HEK293 cells expressing recombinant 5-HT2A, 5-HT2B and 5-HT2C (INI) receptors. Competition binding experiments consisted of addition of 5 μL of serially diluted test compound, 50 μL of radioligand stock diluted in Assay Buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2), and 145 μL of diluted membrane expressing the receptor of interest to 96-well microtiter plates, which were then incubated for one hour at room temperature. Assay incubations were terminated by rapid filtration through Perkin Elmer GF/C filtration plates under vacuum pressure using a 96-well Packard filtration apparatus, followed by washing the filter plates several times with ice cold Assay Buffer. Plates were then dried at 45° C. for a minimum of four hours. Finally, 25 μL of BetaScint scintillation cocktail was added to each well and the plates were counted in a Packard TopCount scintillation counter. In each competition study, test compounds were assayed at 10 concentrations with three replicates at each test concentration. |
| Affinity data for this assay | |
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