Electroformation and Fusion of Giant Lipid Vesicles

Giant unilamellar vesicles made of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and cholesterol (2:1 molar ratio) were prepared following the electroformation method⁸ . A lipid mixture (5 μL, 1 g/L) dissolved in chloroform were spread onto platinum wires mounted in a custom made polytetrafluoroethylene chamber. The lipid mixture was dried with a gentle stream of N₂ and subsequently submerged in a 300 mM sucrose buffer. The wires were connected to a function generator. A 10 Hz 2.0 V sine wave was applied for 1h, with the frequency being reduced to 2 Hz for an extra 30 minutes. Supported lipid bilayers made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3phospho-L-serine (DOPS) (molar ratio 4:3:3) were prepared following the spin coating method. The lipid mixture (25 μL of 1 g/L) dissolved in chloroform:methanol (1:1 volume ratio) were spin-coated (30 s, 3000 rpm) on plasma treated coverslips (#1.5). The coverslips were then mounted on AttoFluor chambers (ThermoFisher), hydrated in HEPES-buffered saline, and cleaned 10 times. The giant vesicles were then transferred to the supported lipid bilayer chamber and after labelling with 200 nM of the exchangeable membrane dye NR4A⁸ and let 15 minutes to settle. To promote membrane fusion 10 mM CaCl₂ dissolved in HEPES-buffered saline were added.