Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

► GUVs made from dry lipid films have wide distributions in lipid composition. ► The main cause of the wide distribution is the demixing of lipids in the dry state. ► A new GUV procedure has been developed to overcome the demixing problem. ► The maximum solubility of cholesterol in diPhyPC lipid bilayer is low (40 mol%). ► Cholesterol interacts unfavorably with the bulky chains of diPhyPC.