We communicated with the technical support division of Biotium and confirmed that Oxazole Gold is identical to SYBR Gold available from other suppliers. Biotium also informed us that the stock solution of Thiazole Green (SYBR Green I) has a concentration of ~10 mM. We preferred to prepare vesicles in the absence of any DMSO which may affect membrane properties, so we attempted to remove DMSO from each commercially available dye stock solution by lyophilization and re-dissolved the residue in Milli-Q water. We found that the SYTO family of dyes could not be re-dissolved in this manner and most of the dye remained stuck to the plastic tube walls. In the case of SYTO 12, when an aqueous solution of the dye was made, the yellow color faded within 30 min, suggesting that the compound may be unstable. Only EvaGreen was commercially available as an aqueous solution (25 µM) and it was concentrated to 0.5 mM. Indolizine dye (Table , Figure ) was soluble in water and stored as a 1 mM stock solution. A few simple mathematical calculations were useful as a guide to choose the concentration of a dye in the hydration solution: (i) if the concentration of a molecule in bulk is 100 µM, there are ~30 molecules inside of a 100 nm vesicle (ii) if the concentration of a molecule in bulk is 10 µM, there are ~3 molecules inside of a 100 nm vesicle. See Figure for structures of dyes where available.