Major-minor mix was done as described in [19 (link), 30 (link)] with few modifications. Mixes of 2 mg/mL of lipids were combined by mol percent: 36.5% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 20% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 30% soy L-α-phosphatidylinositol (PI), 8% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 5% 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 0.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) and 0.015% of DSPE-PEG(2000) Biotin were purchased from Avanti Polar Lipids (Alabaster, AL). When GUVs were prepared with lysophospholipids, 10% soy L-α-lysophosphatidylinositol (LysoPI). The mix was dried under nitrogen flow and resuspended in chloroform.
Lipid mixes were deposited (20 μg) on two indium-tin oxide coated (ITO) slides (10 μg each) and dried for 30min at 30°C in a vacuum chamber. The ITO slides were sealed with each other using a silicone O-ring as spacer, opened to introduce 200μL sucrose buffer (500mM adjusted to 469 mOsm) and later sealed with kwik-cast silicone sealant (Sarasota, FL). GUVs were electroformed (1V, 10Hz) for 2 hr at room temperature (∼23°C). After the electroformation, the chamber was directly placed at 4°C. Eventually, GUVs were pipetted into a siliconized eppendorf tube and stored at 4°C and used within two days.
Lipid mixes were deposited (20 μg) on two indium-tin oxide coated (ITO) slides (10 μg each) and dried for 30min at 30°C in a vacuum chamber. The ITO slides were sealed with each other using a silicone O-ring as spacer, opened to introduce 200μL sucrose buffer (500mM adjusted to 469 mOsm) and later sealed with kwik-cast silicone sealant (Sarasota, FL). GUVs were electroformed (1V, 10Hz) for 2 hr at room temperature (∼23°C). After the electroformation, the chamber was directly placed at 4°C. Eventually, GUVs were pipetted into a siliconized eppendorf tube and stored at 4°C and used within two days.