Measurement of mitochondrial superoxide with MitoSOX red

Mitochondrial superoxide formation was evaluated using MitoSOX red (Molecular Probes, Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, the H9c2 cells were plated at 4.0×10⁵ cells/well in 6-well tissue culture plates, and were treated as described above. Following the indicated incubations, 2.5 µM MitoSOX red was added for 15 min at 37°C. MitoSOX red is a fluorogenic dye for the highly selective detection of superoxide in the mitochondria of living cells and, once targeted to the mitochondria, it is oxidized by superoxide and emits red fluorescence. MitoSOX red is readily oxidized by superoxide, but not by other ROS-generating systems. Cell fluorescence was measured using fluorescence-activated cell sorting (FACS, BD Biosciences) by counting 10,000 events/min, and analyzed using CellQuest software, (version 5.2.1). These data are reported as percentages of positive cells, as referred at the 'gate', which was the sequential identification and refinement of the cellular population using MitoSOX red as the marker that was visualized by fluorescence as a unique emission spectrum.