Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues.
BACKGROUND
Chloromethyl-X-rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM).


METHODS
Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow analysis of endogenous NADH fluorescence. The sensitivity of MTG fluorescence to H(2)O(2) was also evaluated using flow cytometry.


RESULTS
CMXRos fluorescence was dependent on MMP, whereas MTG fluorescence was not affected by MMP, using either flow or LSCM. Specific staining of mitochondria was seen with both dyes in all cell types tested, without evidence of cytotoxicity, as determined by NADH levels. H(2)O(2) damage slightly increased MTG staining of cells.


CONCLUSIONS
Our results indicate that CMXRos is a nontoxic sensitive indicator of relative changes in MMP, whereas MTG is relatively insensitive to MMP and oxidative stress, using both flow and LSCM analyses, provided optimal staining conditions are used. In addition, these dyes can be useful for the study of mitochondrial morphology and function in whole tissues, using LSCM.
Pendergrass
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W
Department of Pathology 357470, University of Washington Medical School, Seattle, WA 98195, USA. pendergr@u.washington.edu
Wolf
N
N
Poot
M
M
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D007834:Lasers; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D009930:Organic Chemicals
D000447:Aldehydes; D004396:Coloring Agents; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C111472:mitotracker green FM; C107472:red dye CMXRos; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D006861:Hydrogen Peroxide