Live Imaging of Synaptic Vesicle Dynamics

PC12 cells were transfected twice with siRNA and with VMAT2-pHluorin, as well on day 1 before imaging, as previously described (Asensio et al., 2010 (link); Onoa et al., 2010 (link)). Glass coverslips containing the transfected PC12 cells were mounted in the perfusion chamber (Warner Instruments) of an inverted Nikon TE3000 microscope fitted with a 60× water objective, and the cells were imaged in modified Tyrode’s solution at room temperature with stimulation by 90 mM K⁺ and alkalinization of the quenched intracellular fluorophore with 10 mM NH₄Cl. Cells were illuminated using a Xenon lamp (Sutter Instruments) with 470/40-nm excitation and 525/50-nm emission filters. Images were acquired every second using a QuantEM charge coupled device camera (Photometrics). MetaMorph software was used to control data collection and perform analysis offline.