Figure 6

Oxidative activity is high in lysosomes that are weakly stained by the acidotropic dye LysoTracker Red DND-99. Cell monolayers were incubated with 200 μg/mL OxyBURST Green H₂HFF BSA at 37°C for 2 hours, washed, and then incubated in the fresh medium containing 200 nM PMA at 37°C for 0.5 hour. Cell monolayers were washed and then incubated with 25 nM LysoTracker Red DND-99, at 37°C for 10 min, to label acidic lysosomes. As indicated in the two upper panels, high OxyBURST fluorescent intensity and thus high oxidative activity are observed in a subset of lysosomes which contain weak fluorescence with the LysoTracker Red and hence low acidity (indicated by arrows). On the other hand, lysosomes strongly stained by the LysoTracker Red only exhibit lower oxidative activities (see arrowheads). The lower panel is the resulting ratio image of the OxyBURST over LysoTracker Red fluorescent intensities, which represents the spatial distribution of an arbitrary oxidative activity/alkalinity ratio in the same cell. Bar, 10 μm.