| Assay Method Information | |
| | 5-HT2A Receptor Binding Assay |
| Description: | ValiScreen Serotonin 5-HT2A (human) cell line (product No: ES-313-C) grown in DMEM/F12 media augmented with 10% FBS, 4 mM GlutaMAX, 0.4 mg/mL Geneticin, 1% Penicillin-Streptomycin were utilized to prepare 5-HT2A membrane fractions. Cells were grown in a 150 mm culture dishes and were harvested at between 70-90% confluency between passages 5-15. Cells were detached and lysed at room temperature with 1 mM HEPES buffer containing 2 mM EDTA at pH 7.4 and homogenized with a hand-held homogenizer. The lysate was then centrifuged (30 minutes at 30,000×G at 4° C.). The resultant pellet was resuspended in a storage buffer (20 mM HEPES, 10 mM MgCl2, 0.1 mM EDTA, pH 7.4 at room temperature) and the suspension frozen and stored at −80° C. until use. Protein concentration was determined via the Bradford method using Coomassie protein assay reagent (Sigma, USA) with Bovine Serum albumin (Sigma, USA) as standard. Aliquots were resuspended in 10 mM HEPES immediately before the experiment.Suspensions of 10 mM HEPES buffer (pH 7.4 at room temperature) containing 10 μg/mL protein, 1 nM (+)-[3H]-ketanserin (Perkin Elmer NET 1233, unlabeled competitor at various concentrations or 10 μM ketanserin for nonspecific binding in a total volume of 500 μL in a 96 well plate. Plates were then incubated in the dark while mixing on a mechanical rocker for 2 h at 37° C. Each plate also contained a dose response curve for ketanserin as a positive control.Following incubation, membrane fractions were collected by vacuum filtration using a Unifilter-96 Cell Harvester (Perkin Elmer) over presoaked UniFilter-96 GF/C P Microplates (Perkin Elmer) and filters were washed with room temperature 10 mM HEPES buffer (pH 7.4 at room temperature) (3×1 mL). The filter plates were dried overnight in a fume hood and the trapped tritium trapped measured via liquid scintillation counting with MicroScint-O (Perkin Elmer), using a MicroBeta2 Plate Reader with 6-detectors scintillation counter (Perkin Elmer) at 55% efficiency. |
| Affinity data for this assay | |
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