Betaplate 1205 liquid scintillation counter

This assay was adapted from previously described studies^{58 (link)}. Briefly, HEK cells expressing the human 5-HT_2A receptor (HEK-h5HT2A) or human 5-HT_2C receptor (HEK-h5HT2C) were used. The cells were grown until confluency on 150 mm plates. Cells were washed with PBS, scraped into 2 mL PBS, and stored at −20°C until use. On the experimental day, the cell suspension was thawed and 10 mL assay buffer (50 mM Tris, pH 7.4 at 37°C, with 0.1% ascorbic acid and 5 mM CaCl₂) was added. This was followed by homogenization via polytron for 5 sec. The homogenate was centrifuged at 15500 rpm for 20 min. To minimize the residual 5-HT concentration, the pellet was resuspended in buffer, homogenized by polytron, and centrifuged as above. The final pellet was resuspended in 10 mL buffer/plate. For the radioligand binding studies, the binding assay included 50 µL BrMeI, 5-HT or buffer, 50 µL cell homogenate, 25 µL [¹²⁵I]DOI (~0.05 nM) and buffer (final volume = 250 µL). Specific binding was defined as the difference between total binding and binding in the presence of 10 µM 5-HT. The reaction was incubated for 1 hr at 37°C and terminated by filtration thru Perkin Elmer A filtermats presoaked in 0.05% polyethylenimine using a Tomtec 96-well harvester. Remaining filter radioactivity was counted via a Perkin Elmer Betaplate 1205 liquid scintillation counter.

HEK cells expressing the human 5-HT_1A receptor (HEK-h5HT1a) were used. Cells were grown to confluence on 150 mm plates in DMEM containing 10% FCS (HyClone), 0.05% penicillin/streptomycin, and 300 µg/mL G418. Cells were collected via scraping into phosphate-buffered saline (PBS) and centrifuged at 270 x g for 10 min. The cell pellet was subsequently homogenized in 50 mM Tris-HCl (pH 7.7) with a polytron, and centrifuged at 27000 × g. The homogenization and centrifugation were repeated to wash any remaining serotonin (5-HT) from the growth media. The final pellet was resuspended at 0.5 mg protein/mL in assay buffer (25 mM TrisHCl, 100 µM ascorbic acid, 10 µM pargyline, pH 7.4). The assay was performed in triplicate in a 96-well plate. The reaction mixture contained BrMeI, 100 µL of cell homogenate (0.05 mg protein/well) and 100 µL of [³H]8-OH-DPAT (0.5 nM final concentration, 170 Ci/mmol, Perkin Elmer) in a final volume of 1 mL. Plates were incubated at room temperature for 60 min and then filtered through polyethylenimine-soaked (0.05%) filters on a Tomtec cell harvester. Filters were then washed with cold 50 mM Tris buffer (pH 7.7) for 6 sec, dried, spotted with scintillation cocktail, and counted for 2 min after a 4 hr delay on a Perkin Elmer Betaplate 1205 liquid scintillation counter (Perkin Elmer). Nonspecific binding was determined with 1.0 µM dihydroergotamine.