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.
Kweon
S M
SM
Biomolecule Research Team, Korea Basic Science Institute, Taejon.
Kim
H J
HJ
Lee
Z W
ZW
Kim
S J
SJ
Kim
S I
SI
Paik
S G
SG
Ha
K S
KS
D016475:3T3 Cells; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006651:Histocytochemistry / Q000379:methods*; D007424:Intracellular Fluid / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000032:analysis*; D013997:Time Factors; D008625:Tiopronin / Q000494:pharmacology; D014966:Xanthenes
D005456:Fluorescent Dyes; D008246:Lysophospholipids; D017382:Reactive Oxygen Species; D014966:Xanthenes; C121372:mitotracker orange; D016718:Arachidonic Acid; D008625:Tiopronin; D002374:Catalase