Extrusion kit

Lipids (~30 mM) were stored in chloroform/methanol (2:1) stock solutions. Lipid films were prepared by drying appropriate amounts of the stock solution in a stream of dry nitrogen followed by vacuum desiccation for at least 2 h to ensure removal of organic solvent. To rehydrate the lipid films, appropriate buffer solutions were added (20 mM NaOAc, 100 mM NaCl, pH 5 and 20 mM MOPS, 100 mM NaCl, pH 7) and vortexed at least 3 times for 60 s. The multilamellar vesicle solution was extruded 30 times through a 50-nm diameter polycarbonate membrane (Whatman, GE Healthcare) using an extrusion kit (Avanti Polar Lipids, Inc.). Vesicle sizes (average hydrodynamic radius ~ 40 nm) were determined by dynamic light scattering using Dynapro NanoStar (Wyatt). Each measurement contained ten 5-s acquisitions and globular proteins were used as the model for calculating the radius with regularization fit. Aqueous lysolipid solutions were prepared from 200 mM stock solutions by dissolving dry powder in working buffers. CMC determined using DLS under our solution conditions are 0.6 mM and 0.8 mM for LPG and LPC, respectively. All buffers were filtered through a 0.22 µm filter (Millipore) before use.

Lipids were stored in organic solvent as described above. Mixed multilamellar vesicles were prepared by drying appropriate amounts of the stock solution in a stream of dry nitrogen. To avoid lipid demixing, this drying process was carried out rapidly at a slightly elevated temperature (~50°C). Subsequently, samples were kept in a high vacuum oven overnight to remove last traces of organic solvent. The lipid mixtures were re-suspended in the appropriate buffer solution. The multilamellar vesicle solution was extruded 30 times through a 100 nm diameter polycarbonate membrane (Avestin, Ottawa, ON) using an extrusion kit (Avanti Polar Lipids, Alabaster, Al). The size of the obtained unilamellar vesicles was checked using dynamic lighting scanning (DLS) (HPPS and Zetasizer, Malvern Instruments, Southborough, MA).