Different methods to estimate the plasma membrane potential difference (PMP) of yeast cells with fluorescent monitors were compared. The validity of the methods was tested by the fluorescence difference with or without glucose, and its decrease by the addition of 10 mM KCl. Low CaCl2 concentrations avoid binding of the dye to the cell surface, and low CCCP concentrations avoid its accumulation by mitochondria. Lower concentrations of Ba2+ produce a similar effect as Ca2+, without producing the fluorescence changes derived from its transport. Fluorescence changes without considering binding of the dyes to the cells and accumulation by mitochondria are overshadowed by their distribution between this organelle and the cytoplasm. Other factors, such as yeast starvation, dye used, parameters of the fluorescence changes, as well as buffers and incubation times were analyzed. An additional approach to measure the actual or relative values of PMP, determining the accumulation of the dye, is presented.
Fig. 1SFluorescence changes of DiSC3(3) added to non-starved yeast cells; effects of anaerobiosis and CCCP. Incubation: 10 mM MES-TEA buffer, pH 6.0, with (black lines) and without glucose (gray lines); effects of H2O2 (17 μmoles) and CCCP (10 μM). The experiment was carried out as described for Fig. 1, and both additions are indicated in the figure. AU, arbitrary units (JPEG 43 kb)