The source of the difference in cholesterol concentrations between GUVs formed by electroformation and those formed by gentle hydration may be a result of the alternative current applied during electroformation. While the phospholipids present in the ternary mixture are zwitterionic and potentially more affected by the alternating current applied during electroformation, cholesterol is neutral, and therefore may be less responsive to the applied current. This would result in lower incorporation into electroformed GUVs. However, the experiments performed here cannot provide a definitive explanation or mechanism for the difference in cholesterol concentrations between the two methods.