| Assay Method Information | |
| | Serotonin transporter (SERT) Uptake Inhibition Assay |
| Description: | Serotonin transporter (SERT) Uptake Inhibition: Compound activity was assessed using an in vitro assay. Briefly, HEK cells expressing recombinant SERT were plated at 50,000 cells/well on a 96 well plate pre-coated with Matrigel one day prior to the experiment. Culture medium (DMEM/FBS) was removed from each well, and 30 μl of assay buffer (Tris-HCl 50 mM, EDTA 4 mM, BDA 0.1%) with the desired concentration of test compound was added. The plate was incubated at 37° C. for 15 minutes. Assay buffer (30 μl) containing the same concentration of compound diluted in [3H]-Serotonin uptake buffer (final concentration 10 nM) was added to each well and the plate was incubated at 37° C. for 5 minutes. The reaction mixture was removed, and the cells were washed (2×) with 100 μl ice-cold assay buffer. Lysis buffer (50 μl) was added to the cells followed by a 5 minute incubation with gentle shaking at room temperature. The lysate was transferred to a 96 well isoplate. Optiphase supermix (100 μl) was added to each well and was thoroughly mixed. Radioactivity was counted with Microbeta Counter and reported as counts per minute (CPM). Fluoxetine was used as a control to measure non-specific uptake and for data normalization. Percent inhibition of [3H]-Serotonin uptake is based on the calculation: 100×(1−(CPMtest sample−CPMnon-specific uptake)/(CPMMAX−CPMMIN)). |
| Affinity data for this assay | |
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