Liposofast extrusion system liposofast

The lipids DPPC and DPPS were used in the preparation of the DPPC liposomes and DPPC:DPPS liposomes with 5, 10 and 15% of DPPS (molar ratio). Lipids were dissolved in chloroform and dried under nitrogen flow. The resulting lipid film was kept under vacuum overnight and suspended in 50 mmol/L Tris–HCl buffer, pH 7.5, containing 2 mmol/L MgCl₂ and 200 μM EGTA, obtaining a final solution of 1.5 mg/mL (DPPC:DPPS). The mixture was then incubated for 1 h at 70 °C, above the critical phase transition temperature of the lipid, and vortexed at 10 min intervals. Large unilamellar vesicles (LUVs) were prepared by submitting the suspension to extrusion through 100 nm polycarbonate membranes in a LiposoFast extrusion system (Liposofast, Sigma–Aldrich). LUVs were prepared and used in the same day as described by Bolean et al. [7 (link)] and [28 (link)].

The lipids DPPC and DPPS were used in the preparation of the DPPC-liposomes and 9:1 DPPC:DPPS-liposomes (molar ratio) as previously described [18 (link)]. Lipids were dissolved in chloroform and dried under nitrogen flow. The resulting lipid film was kept under vacuum overnight and suspended in the stock buffer (50 mM Tris-HCl buffer, pH 7.5, containing 2 mM MgCl₂), to obtain a final lipid solution of 1.5 mg/mL. The mixture was then incubated for 1 h at 70 °C, above the critical phase transition temperature of the lipid, and vortexed at 10 min intervals. Large unilamellar vesicles (LUVs) were prepared by submitting the suspension to extrusion through 100 nm polycarbonate membranes in a LiposoFast extrusion system (Liposofast, Sigma-Aldrich). Liposomes also were labeled with 0.2 mol % of fluorescent Rhodamine 6G, to facilitate detection by microscopy and flow cytometry and to allow time-lapse observations via confocal microscopy.