Figure 9

Heterogeneous distribution of oxidative activity in lysosomes is not due to heterogeneous distribution of the OxyBURST Green H ₂ HFF BSA Cell monolayers were grown on circular glass coverslips and labeled with OxyBURST Green H₂HFF BSA as described. The cells were then mounted onto a temperature control stage with a perfusion chamber for microscopy. The temperature was controlled at 37°C throughout the experiment. Cells were then treated with 200 nM PMA for 30 min, followed by 25 nM LysoTracker Red DND-99 for 5 min. Cells were then washed with PBS and the fluorescence images of both oxidized OxyBURST Green H₂HFF BSA and LysoTracker Red were acquired. As shown in the panel A, oxidation activity is high in a subset of lysosomes (a, see arrow), which accumulate little LysoTracker Red (b). To oxidize all the OxyBURST Green H₂HFF BSA in the same cells, H₂O₂ was added to the chamber to a final concentration of 0.05% in PBS. The cells were then incubated for an additional 15 min to allow oxidation of all the lysosomal OxyBURST Green H₂HFF BSA. Afterwards, the fluorescent image of oxidized OxyBURST Green H₂HFF BSA from the same cell was then recorded as shown in (c). Bar, 10 μm.