Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy - PubMed (original) (raw)

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Partha Mukhopadhyay et al. Nat Protoc. 2007.

Abstract

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

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Figures

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.

Figure 2

Determination of mitochondrial superoxide production measured by flow cytometry and confocal microscopy using MitoSOX. (a) Complex III inhibitor Antimycin A (AntA, 20 μM) time-dependently increases mitochondrial-derived superoxide generation measured by flow cytometry. There is no significant change in the mean fluorescence intensity of vehicle-treated cells (upper row, purple histograms and trace), whereas AntA shifts histograms to the right, thereby increasing the mean fluorescence intensity of MitoSOX (lower row, red histograms and trace). For each histogram, the x axis shows % cells and the y axis shows the FL2 channel (Mitosox Red). (b) Time-lapse images of Mito-GFP-transfected MPMVECs incubated with the mitochondrial O2•−-sensitive fluorescent dye MitoSOX Red (10 μM) before and after the addition of the mitochondrial complex III inhibitor AntA (2 μM). As shown, the representative cell expresses Mito-GFP (green fluorescence) and has very low MitoSOX fluorescence (red color) before stimulation. AntA triggers time-dependent increase in mitochondrial superoxide formation (increasing red fluorescence, which is colocalized with green Mito-GFP fluorescence; see yellow color in overlaid images). (c) Representative tracings of Mito-GFP and MitoSOX Red fluorescence in response to AntA (2 μM) showing increased MitoSOX Red fluorescence in stimulated cells. (d) MitoSOX Red fluorescence is unchanged in unstimulated cells. F.A.U., fluorescence arbitary unit.

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Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy - PubMed (original) (raw)