Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress

Abstract

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

Refinement of the Dichlorofluorescein Assay for Flow Cytometric Measurement of Reactive Oxygen Species in Irradiated and Bystander Cell Populations

“…2B). Although substrate diffusion is likely dependent on concentration and cell type, the DCFH-DA loading kinetics measured here correlates well with previous reports that intracellular DCFH-DA concentration stabilized after 10 min in cardiomyocytes (39) and 15 min in endothelial cell cultures (29), and that the concentration remained stable for over 1 h. To avoid artifacts, cells in suspension with DCFH-DA should not be exposed to radiation prior to substrate saturation.…”

Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats

“…The 2-fold increase in DCF fluorescence of PMNs isolated from iron-deficient rats indicates that oxidants are increased, possibly because of mitochondrial oxidant production (34). A plausible mechanism is that the lack of heme-containing electron-transport proteins (18) such as cytochrome c oxidase (complex IV) and͞or iron sulfur clusters in the mitochondria promotes oxidant leakage (30) (see below).…”

Anthracycline-induced phospholipase A2 inhibition

“…To visualize the involvement of free radicals in doxorubicin-mediated myocyte injury we used confocal laser scanning microscopy and the oxidant-sensitive fluorescent dye, 2′,7′-dichlorofluorescin. Exposure to doxorubicin produced a rapid and concentration-dependent increase in the fluorescence that appeared to occur in close proximity to the mitochondria [6,7]. We have also shown that pretreatment of the cells with dexrazoxane inhibited doxorubicin-induced increase in oxidant-sensitive fluorescence.…”