For monitoring synaptic vesicle release, human iPS cell derived neurons were loaded with 10 μM FM1-43 for 2 min in FM image buffer (FMIB, consisting of 170 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.2 mM MgCl2, 1.3 mM CaCl2, 5 mM glucose, 20 mM N-tris (hydroxymethyl)-methyl-2-aminoethane-sulfonic acid, pH7.4, ~360 mOsmol) supplemented with 60 mM KCl, followed by a wash with 10 mM FM1-43 in FMIB for 2min. Subsequent washing was with FMIB for 5 min, followed by FMIB supplemented with 1 mM ADVASEP-7. FM1-43 imaging was performed on a Nikon TE2000 imaging system with a 20× objective. Neurons were perfused with FMIB for1 min as the baseline, followed by stimulation with 60 mM KCl for 4 min. Cells were excited at FITC spectra, and the green fluorescence was collected as FM1-43 signal. Images were acquired every 5 s and analysed using NIH ImageJ software. The FM1-43 signal was determined by F [F = (F1−B1)/(F0−B0)], which was normalized to the mean fluorescence intensity measured at the baseline condition (set as 1).
