cDICE method produces giant lipid vesicles under physiological conditions of charged lipids and ionic solutions

Giant unilamellar vesicles are a powerful and common tool employed in biophysical studies of lipid membranes. Here we evaluate a recently introduced method of vesicle formation, continuous droplet interface crossing encapsulation (cDICE). This method produces monodisperse giant unilamellar vesicles of controlled sizes and high encapsulation efficiencies, using readily available instrumentation. We find that mixtures of phospholipids within vesicle membranes produced by cDICE undergo phase separation at the same characteristic temperatures as lipids in vesicles formed by a complementary technique. We find that the cDICE method is effective both when vesicles are produced from charged lipids and when the surrounding buffer contains a high concentration of salt. A shortcoming of the technique is that cholesterol is not substantially incorporated into vesicle membranes. The cDICE method produces giant unilamellar vesicles composed of mixtures of phospholipids, even when the lipids are charged and when the surrounding buffer contains high salt. However, cDICE does not incorporate substantial cholesterol into membranes.