We used EZ-C1 viewer (Nikon, version 3.50) to measure the diameter of liposomes and the fluorescence intensity of their interiors, their membranes and the surrounding solution as a function of time. We determined that the fluorescence intensity was linearly related to the concentration of Rho123 in solution (see Supporting Information, Fig. S2 ). To overcome any experimental variation, we performed a two point calibration before each experiment to determine the Rho123 concentration from fluorescence intensity values using the average intensity inside GUVs without P-gp at the starting time (concentration of Rho123 ~ 0 µM) and the average intensity of the solution outside of GUVs at the starting time (concentration of Rho123 = 1 µM). For the analysis of Ps and kT, we omitted liposomes that were clearly not unilamellar or that visibly contained small vesicles inside (see Supporting Information, Fig. S5 ), as well as liposomes whose fluorescence increase did not follow the model represented by Eq. (2) (i.e. R2 < 0.9). The time-dependent fluorescence increase (and hence the increase in Rho123 concentration) of most liposomes could, however, be described very well with Eq. (2) (R2 > 0.98).
We used a non-parametric Kruskal–Wallis test in Origin 8.0 to assess the statistical significance of the data for each experimental condition.
We used a non-parametric Kruskal–Wallis test in Origin 8.0 to assess the statistical significance of the data for each experimental condition.