Preparation of giant unilamellar vesicles (GUVs)

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 25 mg/ml) or 1,2 Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC, 25 mg/ml) from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) were dissolved in chloroform (Sigma-Aldrich, Bornem, Belgium) to obtain 20 mM stock solution, which were further diluted with chloroform to final working concentration of 10 mM. The stock of cholesterol (1 mM) (Sigma-Aldrich) was also prepared in chloroform. We produced GUVs with incorporated fluorescent probes (Laurdan and DPH) using a 1/800 probe-to-lipid ratio. GUVs were prepared by the electroformation method (hydration of a dry lipid film in an oscillating electric field) using a Nanion Vesicle Prep Pro setup (Nanion Technologies GmbH, Munich, Germany). Lipid solutions (40 µl) containing 10 mM DPPC or DPhPC, with or without fluorescent probes, or 10 mM DPhPCs with 20% cholesterol (with or without fluorescent probes) were deposited on the conductive side of indium tin oxide (ITO) coated glass electrode and evaporated under vacuum to remove chloroform traces. After total solvent evaporation, a greased O-ring was placed around the dried lipid film and filled with 1 M sorbitol solution, pH 7.0. Then, a second ITO-electrode was placed on the ring. GUV formation was induced by application of an alternating voltage of 3 V peak-to-peak for 2 h with a frequency of 5 Hz, while keeping the temperature at 60 °C (DPPC GUV) or 37 °C (DPhPC GUV). Formation of GUV was followed by visualization with an upright bright field microscope (amplification 40X). Last, GUVs were plated into flat-bottom 96-well microtiter plates (Greiner Bio-One), and treated with LPS extracted from E. coli (serotype 0127:B8, Sigma-Aldrich) or S. minnesota (Sigma-Aldrich) or the fluidizing agent benzyl alcohol (Sigma-Aldrich).