MitoSOX Red is a novel fluorogenic dye recently developed and validated [11
] for highly selective detection of superoxide in the mitochondria of live cells. Numerous recent studies utilizing different stimuli of superoxide production coupled with fluorescent microscopy have demonstrated detectable changes with MitoSOX in mitochondrial superoxide generation in olygodendrocytes [11
], retinal ganglion cells [15
], neurons [16
], isolated cardiomyocytes [18
], and parasite Trypanosoma cruzi
However, the disadvantage of the fluorescent microscopy technique is that it allows measurements of various biological processes only in a relatively limited number of cells simultaneously, and is semiquantitative. In addition, cells loaded with fluorescent probes during the fluorescent microscopy receive more exposure of lasers or UV, which can increase ROS generation by itself, than during the flow cytometry experiments.
In the present study, we demonstrate using well-established stimuli of mitochondrial superoxide and ROS production such as mitochondrial complex III inhibitor Antimycin A [11
], herbicide paraquat [11
], chemotherapeutic agent Doxorubicin (known for its cardiotoxicity; [21
]) and high glucose [23
], a marked ~3–7 fold dose-and time-dependent augmentation of mitochondrial superoxide generation measured by increased fluorescent intensity of MitoSOX by flow cytometry. In these experiments the mitochondrial superoxide generation measured by MitoSOX could largely be prevented by pretreatment of cells with high concentrations of cell permeable SOD (data not shown) as previously described [11
]. These results are also in well-agreement with previous reports demonstrating increased superoxide and/or other ROS generation either in isolated mitochondria exposed to these pathologically relevant stimuli or in cells loaded with cytosolic ROS detecting probes ([20
]; in most of the later experiments the mitochondrial contribution to ROS/superoxide production was deduced from the use of various mitochondrial uncouplers (known also to dissipate mitochondrial membrane potential)), and with a recent fluorescent microscopy study using MitoSOX [11
] along with the confocal microscopy data shown in the present manuscript.
One possible limitation of the detection of mitochondrial superoxide production using MitoSOX is that it can bind to the nuclear DNA following oxidation. This could be minimized with individual optimization of loading conditions for each cell type and normalization of the data to the control cells loaded with MitoSOX. Another possible limitation is that under various conditions, where increased cytosolic superoxide generation may also be enhanced in addition to the mitochondrial (and is already present at a time of the loading with MitoSOX) some of the MitoSOX may be oxidized during the transport from cell membrane before entering into the mitochondria. In theory, superoxide produced from the mitochondria can also diffuse to and present in the cytosol contributing to similar scenario during the loading. However, as clearly demonstrated by our chronic treatment protocols with high glucose and Doxorubicin in endothelial cells and/or myocytes (supported by confocal microscopy data), this level of cytosolic oxidized MitoSOX is likely to be negligible in comparison with the increase in fluorescence detected in the mitochondria.
Collectively, these results establish a new method allowing simple, selective and quantitative detection of the mitochondrial superoxide generation simultaneously in a large number of live cells by flow cytometry, which can easily be applicable for virtually any cell types. The clear advantages of this method over other cell-based techniques and fluorescent microscopy are: tremendous speed, exquisite precision, and possibility of simultaneous quantitative measurements of multiple cellular parameters with maximal preservation of cell viability and cellular functions.