Fig. 5. LysoSensor Green DND-189 is a vital dye that fluoresces in the acidic compartments of the yeast cell. (A) Comparison of LysoSensor Green DND-189 and Quinacrine staining. Wild-type cells were stained separately with LysoSensor Green DND-189 and Quinacrine as described in Materials and methods. (B) Wild-type cells were incubated at 0 °C for 30 min with 30 µmol l^-1 FM 4-64 in YPD medium buffered with 100 mmol l^-1 Hepes, pH 7.6. The cells were harvested by centrifugation, washed once with the same buffer, and resuspended in 0.35 ml of fresh medium containing 4 µmol l^-1 LysoSensor Green DND-189. Cells were then incubated for 5 min, harvested by centrifugation, washed and resuspended in YPD medium. Portions were removed for microscopy and photographed at 25 min (top) and 45 min (bottom). Top left of each panel, FM 4-64 fluorescence; top right, the same cells in visible light; bottom left, LysoSensor Green DND-189 fluorescence; bottom right, the double-labeled image. The images were recorded, merged and processed using the Zeiss LSM Image Browser.