| Assay Method Information | |
| | Characterization of Antagonism at Human 5-HT2A Receptors using Fluorometric Ca2+Detection |
| Description: | CHO-K1 cells expressing human recombinant 5-HT2A receptors and Gα16 (purchased from Euroscreen Fast, Brussels, BE) in culture were cryopreserved according to established protocols, using 90% FBS/10% DMSO as medium. Prior to the experiment, cells were thawed, resuspended in PowerCHO™ 2 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antimycotic solution, and 1% pyruvate. Cells were seeded in 96-well microplates at a density of 40,000 cells/well and incubated overnight at 37° C. in a humidified atmosphere containing 5.0% CO2. On the day of the experiment, plates were washed with assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, 2 mM probenecid, pH 7.4) using a plate washer (Elx405UCWS, Biotek, Winooski, VT, USA), then 50 μL/well of 4 μM Fluo-4 AM (Thermo Fisher Scientific) in assay buffer was added. After dye loading (60 minutes, 37° C., in darkness), plates were washed with assay buffer using the plate washer leaving 50 L/well residual volume, then 50 L/well assay buffer containing vehicle (3% DMSO in assay buffer) or test compounds (3× of the final concentration) were added and the cells were incubated for an additional 10 minutes at 37° C. |
| Affinity data for this assay | |
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