After drying the lipid film, a small magnetic stir bar is placed into a flask, the lucigenin solution is added (0.5 mL), and the flask is sealed with a closed vacuum adapter (or stopper). The flask is then sonicated for approximately 30 seconds to obtain a homogenous mixture (vortexing could also be used). No more lipids should be stuck on the wall of the flask after the sonication. The mixture is further stirred for 1 hour on a magnetic stirrer to produce heterogeneous vesicles (Figure ).