GUV preparation

GUVs were prepared by electroformation on Pt wires (20). 1,2-dioleoyl-sn-glycero-3-phosphocholine was dissolved in chloroform/methanol (36:1, v/v) mixture at a final concentration of 0.06 mg/mL. Approximately 30 μL of the solution was deposited and dried on two Pt electrodes (40 mm long, 0.3 mm diameter). Solvent traces were removed in vacuum (90 min). Next, the electrodes were connected to 10 Hz, 0.15 V_pp alternating voltage and were placed into a 1 × 10 × 45-mm cuvette filled with a 150 mM aqueous sucrose solution. The distance between the two wires was 4 mm. The voltage amplitude was gradually increased from 0.15 to 2.1 V during 30 min and was left at 2.1 V for another 90 min. Finally, to promote vesicle closure and detachment from the electrodes, the frequency was gradually reduced to 1 Hz in ∼20 min. The described method led to formation of 1–10-μm diameter GUVs that were then stored at 4°C.