Master Stock Solutions. Master stock solutions were prepared with the lipid mixture of interest dissolved in chloroform in high enough quantities such that multiple batches of GUVs could be produced from the same vial. Master stocks were made by first adding several hundred microliters of chloroform to a 2 mL glass vial. Lipids were then added to the vial by withdrawing the appropriate volume from a pure lipid stock solution in chloroform and then injecting the volume beneath the chloroform in the master stock vial. This ensured that each lipid remains fully dissolved within the master stock. All master stocks also contained 0.1 mol % TR-DHPE so GUV patches could be examined via fluorescence microscopy once ruptured on pattered substrates. Critically, any comparison between different methods of GUV formation was done using lipid films produced from one of these master stocks.