Staining of cellular mitochondria with LDS-751.
We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 microg/ml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing.
Snyder
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Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th St. Hamilton, MT 59840, USA. ssnyder@NIAID.NIH.gov
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D000818:Animals; D001152:Arsenicals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002460:Cell Line; D004072:Digitonin; D005456:Fluorescent Dyes; D008564:Membrane Potentials; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000648:ultrastructure*; D009000:Monocytes / Q000648:ultrastructure; D009930:Organic Chemicals; D020112:Rhodamine 123; D013194:Staining and Labeling / Q000379:methods*
D001152:Arsenicals; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C029341:oxophenylarsine; C057718:6-(Dimethylamino)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)-1,3-butadien-1-yl)-1-ethylquinolinium perchlorate; D020112:Rhodamine 123; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004072:Digitonin