Preparation of Lipid Vesicles by Extrusion

LUVs were made by extrusion. Lipid solutions containing 12.5 μmole of lipids were made using a 4:1:0.025 molar ratio of POPC:POPG:Ch or a 5:0.025 molar ratio of POPC:Ch, expect where indicated and as previously described13 (link). In brief, the lipid solution was dried under a stream of argon gas, residual chloroform was then removed by storage for 1 hr under vacuum. Lipid films were then dissolved in 500 μl of potassium buffer (10 mM K₂HPO₄, 50 mM K₂SO₄, 5 mM MOPS, pH 7.4) and hydrated for 30 min at 5 °C above the phase transition temperature of the lipid mix with vortex mixing every 5 min. Lipid solutions were then freeze-thawed 15 times transferring between a dry ice ethanol bath and a water bath at hydration temperatures before extrusion using an Avanti Mini-Extruder, 26 times with 100 nm pore-size membranes (Whatman Nuclepore Track-Etched Membranes, GE Healthcare Bio-Sciences, PA, USA) at hydration temperatures. LUVs were stored at 4 °C and used within one week.