2.2.2. Preparation of Large Unilamellar Vesicles

Fluorescence spectroscopy of 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), 1,6-diphenyl-1,3,5-hexatriene (DPH) and Di-8-ANEPPS is performed on large unilamellar vesicles (LUVs). LUVs are formed by means of the extrusion method as described in [26]. The lipid and the indicated fluorescent probe are dissolved and mixed in chloroform/methanol (1:1 v/v). Laurdan or DPH are mixed with the lipids in the initial organic solution at 1:200 probe:lipid molar ratio. The fluorescent probe di-8-ANEPPS in ethanol at 1 mg/mL stock solution concentration, is mixed with the lipids in the initial organic solution at 1:250 probe-to-lipid molar ratio. Afterward, the solvent is removed under a stream of oxygen-free dry nitrogen. The residues are subsequently maintained under vacuum overnight, and then filtered (0.2 µm) sugar solution is added at room temperature (22 ◦C) to yield a lipid concentration of 1 mM. The samples are heated at 60◦C for 5 min, vortexed for 1 min, then left in a sonication bath for 1 min, and finally cooled in ice for

5 min. This heating/cooling procedure is repeated three times to ensure the sample homogenization. The multilamellar vesicles obtained at this stage are then extruded with a LiposoFast small-volume extruder equipped with polycarbonate filters (Avestin, Ottawa, Canada) as follows: 11 extrusions through 800 nm, followed by 21 extrusions through 100 nm filters. The final lipid concentration in cuvette is 200 µmol/L. LUV samples are studied the same day after equilibration for 20 min at room temperature (22 ◦C). Each sample is measured 10 times after gentle pipetting and averaged by three different LUV preparations.
