Lysosomal membrane permeabilization (LMP) causes the loss of the pH gradient over lysosomal membranes, and as a result cells show reduced staining with lysosomotropic dyes compared with untreated control cells. (A–E) HeLa cells, untreated or cells treated with 30 nm bafilomycin A1 (v-ATPase inhibitor) for 30 min, or with 5 mm Leu-Leu-O-methylester (LLOMe; a known LMP inducer) for 15 min, were stained with acridine orange or LysoTracker Green and analyzed with a flow cytometer. Before the fluorescence analysis, the gate was set around live cells in a density plot of forward and side scatter (FSC/SSC) to exclude debris. Results of the acridine orange or Lysotracker Green staining are presented as frequency histograms of fluorescence intensity (B,C) and as mean ±s.d. values for the geometric mean of fluorescence intensity (D,E). The experiment was performed in duplicate. (F–I) RAW264.7 cells, stained with acridine orange (F–H), and HeLa cells, stained with Lysotracker Red (I), were analyzed with a confocal laser scanning microscope. Cells stained with acridine orange were analyzed with three different combinations of excitation and emission light. Paired untreated control and LLOMe-treated samples were analyzed at the same imaging settings. Scale bar, 10 µm.