| Assay Method Information | |
| | Norepinephrine transporter (NET) Uptake Inhibition ASsay |
| Description: | Compound activity was assessed using a convenient in vitro assay. Briefly, HEK cells expressing recombinant NET 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 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]-Norepinephrine uptake buffer (final concentration 20 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 with 100 μl ice-cold assay buffer twice. 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 with complete mixing. Radioactivity was counted with Microbeta Counter and reported as CPM. Desipramine was used as a control to measure non-specific uptake and for data normalization. Percent inhibition of [3H]-Norepinephrine uptake calculations: 100×(1−(CPMtest sample−CPMnon-specific uptake)/(CPMMAX−CPMMIN)). |
| Affinity data for this assay | |
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