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. Author manuscript; available in PMC: 2008 Feb 4.
Published in final edited form as: Nat Protoc. 2007;2(9):2295–2301. doi: 10.1038/nprot.2007.327

Figure 1.

Figure 1

Simultaneous determination of apoptotic markers and mitochondrial superoxide formation by flow cytometry and confocal microscopy following DOX treatment. (a) Dose-dependent changes in the number of apoptotic (Annexin V positive) and necrotic (Annexin V and Sytox Green positive) endothelial cells and MitoSOX fluorescence in total (dark green), normal (light green), apoptotic (blue) and dead cells (orange) following DOX treatment for 16 h. (b) Simultaneous live imaging of endothelial mitochondrial superoxide production and apoptosis following DOX treatment. All the three cells shown are Annexin V positive (blue rings). In two early apoptotic cells, MitoSOX signal shows mitochondrial pattern, whereas in one late apoptotic/dead cell (marked with an arrow), there is a strong nuclear but no mitochondrial staining pattern. Note that detailed analysis by flow cytometry of various cell populations also revealed very strong oxidized MitoSOX fluorescence in dead cells (orange histograms), because of the release of the fluorophore from the mitochondria and nuclear binding of MitoSOX Red in dead cells (Fig. 1b, marked cell). Therefore, the dead cells should be excluded from the analysis of the mitochondrial superoxide generation by flow cytometry.