Liposome preparation

Small unilamellar vesicles were prepared to approximate the lipid composition of synaptic vesicles (Takamori et al., 2006), 50 mol% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 30 mol% DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), and 20 mol% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), supplemented with 1 mol% DOPE-Cy5 (Avanti Polar Lipids). Lipids were mixed in chloroform and dried under a mild stream of nitrogen gas. Dried lipids were then lyophilized for 3 h (VWR, Avantor), before being rehydrated in 25 mM Tris-HCl (pH 7.4). Liposomes were formed by 10 consecutive freeze‒thaw cycles (–196°C to 30°C). To form uniformly sized liposomes, the mixture was extruded 20 times through 50 nm-diameter polycarbonate filters (Avanti Polar Lipids). Liposome size distribution was confirmed using dynamic light scattering (average size ±50 nm) and re-extruded when standard deviation >20 nm.