Blue dye

nAChR functional activity was determined by measuring nAChR-induced membrane potential change, which can be directly read by Molecular Devices membrane potential assay kit (Blue Dye) (Molecular Devices, Sunnyvale, CA) using the FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA). The HEK cells with stably expressing α3β4 or α4β2 nAChR were seeded in a 96-well plate (4000 cells per well) 1 day prior to the experiments. For agonist assays, after brief washing, the cells were loaded with 225 μL of HBSS assay buffer (Hank’s balanced salt solution with 20 mM of HEPES, pH 7.4), containing the Blue Dye, and incubated at 37 °C. After 30 min, 25 μL of the appropriate compounds were dispensed into the wells by the FlexStation and nAChR stimulation-mediated membrane potential change was recorded every 3 s for 120 s by reading 565 nm fluorescence excited at 530 nm wavelength. For the antagonist assay, the cells were loaded with 200 μL HBSS buffer containing the Blue Dye and incubated at 37 °C. After 20 min, 25 μL of test compounds was added, and after another 10 min, 25 μL of epibatidine (or nicotine) was added by the FlexStation, to a final concentration of 100 nM, with fluorescence measured as described above. The change in fluorescence represents the maximum response minus the minimum response for each well. GraphPad Prism was used to determine the EC₅₀ and IC₅₀ values.