Heterogeneous distribution of oxidative activity in lysosomes is not due to heterogeneous distribution of the OxyBURST Green H2HFF 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.
Chen BMC Cell Biology 2002 3:21 doi:10.1186/1471-2121-3-21