Quantifying Cholesterol Concentrations. Cholesterol concentration was also examined in GUV patches. Unlike phospholipids, Cholesterol localized significantly to the edges of GUV patches formed by either electroformation or gentle hydration as seen in Figure and Figure . This makes quantification of cholesterol variability in GUVs significantly more challenging as the relative ratio of edge to center within the analyzed region needs to be considered as well as how the overall concentration of cholesterol in a patch may affect its partitioning between the edge and center. As a result, the variability in cholesterol concentration from GUV to GUV was not assessed. Instead, the average cholesterol concentration was determined for different GUV formation methods. Ternary GUV patches were formed via gentle hydration and electroformation using a master stock with nominal composition DSPC:POPC: 2 H31-POPC: 13 C27-CHOL 40:20:20:20. The average concentration was then calculated either including or excluding the cholesterol-rich edges. Regardless of the analysis method used, the average concentration of cholesterol was lower in electroformed GUV patches, as can be seen in Figure . The average 2 H31-POPC concentration was the same between the two methods regardless of the analysis method (Figure ). Average cholesterol concentration was also examined in GUV patches where all three components are isotopically labeled (Figure ). These experiments also demonstrate lower average cholesterol concentrations in electroformed GUVs.