Real-time measurement of intracellular reactive oxygen species using Mito tracker orange (CMH2TMRos)

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH2TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120-150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH2TMRos. Incubation with glucose oxidase, exogenous H2O2, KO2 and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH2TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH2TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H2O2.