Universol

serotonin 5-HT_2B receptor competition binding experiments were carried out in membranes from CHO-K1-5-HT_2B cells prepared in our group. On the day of the assay, membranes were defrosted and resuspended in binding buffer (50 mM Tris-HCl, 4 mM CaCl₂, 0.1% ascorbic acid, pH 7.4). Each reaction well of a 96-well plate contained 5 μg of protein, 1 nM [³H]LSD (83.6 Ci/mmol, Perkin-Elmer, Waltham, MA, USA), and different concentrations of the compounds in the range from 0.01 nM to 10 µM. Non-specific binding was determined in the presence of 50 μM serotonin (Sigma Aldrich, Spain). The reaction mixture was incubated at 37 °C for 30 min, after which samples were transferred to multiscreen GF/C 96-well plates (Millipore, Spain) pretreated with 0.5% polyethylenimine (PEI, Sigma Aldrich, Spain), filtered, and washed four times with 250 μL wash buffer (50 mM Tris-HCl, pH 7.4). The filters were dried, 35 μL Universol (MP Biomedicals, Spain) per well were added and radioactivity was measured as described above.

Serotonin 5-HT_2A receptor competition binding experiments were carried out in membranes from CHO-FA4-5-HT_2A cells prepared in our group. On the day of the assay, membranes were defrosted and resuspended in binding buffer (50 mM Tris-HCl, pH 7.5). Each reaction well of a 96-well plate contained 80 μg of protein, 1 nM [³H]Ketanserin (50.3 Ci/mmol, Perkin-Elmer, Waltham, MA, USA), and different concentrations of the compounds in the range from 0.01 nM to 10 µM. Non-specific binding was determined in the presence of 1 μM methysergide (Sigma Aldrich, Spain). The reaction mixture was incubated at 37 °C for 30 min, after which samples were transferred to multiscreen GF/B 96-well plates (Millipore, Spain) pretreated with 0.5% polyethylenimine (PEI, Sigma Aldrich, Spain), filtered, and washed six times with 250 μL wash buffer (50 mM Tris-HCl, pH 6.6). The filters were dried, 35 μL Universol (MP Biomedicals, Spain) per well were added and radioactivity was detected in a microplate beta scintillation counter (Microbeta Trilux, Perkin-Elmer, Waltham, MA, USA).