The stock solutions of lipids are allowed to warm up to room temperature prior to opening the vials, to avoid water condensation. The adequate lipid solution volumes required to obtain a 0.4 mM final lipid concentration are added into a 5 mL round bottomed flask using pipettes for organic solvents. For example, to prepare 20 mL liposomes with 0.4 mM concentration, 8 µmol of lipids is needed, corresponding to 5.6 µmol of POPC and 2.4 µmol of cholesterol for a POPC/cholesterol 7:3 mixture. The solvent is evaporated by applying a gentle nitrogen or argon flow (bringing lower risks of contamination compared to the use of a rotary evaporator). The dried lipid film (Figure ) is then placed in high vacuum for at least 1 hour, but this could also be done overnight. Ideally, the lipid film should be protected from UV light.