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Cytometry. 1999 Apr 1;35(4):311-7.

Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry.

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1
Department of Pathology, University of Washington, Seattle 98195-7705, USA. mpoot@u.washington.edu

Abstract

BACKGROUND:

The possible relationships between changes in mitochondrial membrane potential and other mitochondrial functions during apoptosis remain controversial.

METHODS:

To detect concomitant changes in mitochondrial function during apoptosis, we performed correlated multiparameter flow cytometry after simultaneous cell staining with several dyes.

RESULTS:

After camptothecin treatment, nonapoptotic cells exhibited a concomitant rise in mitochondrial membrane potential [8-(4'-chloromethyl) phenyl-2, 3, 5, 6, 11, 12, 14, 15-octahydro-1H, 4H, 10H, 13H-diquinolizino-8H-xanthylium chloride, or CMXRos; CMXRos fluorescence divided by MitoTracker Green fluorescence], NADH level (ultraviolet-excited blue autofluorescence), and oxidative turnover (H2-CMXRos oxidation). Frankly apoptotic cells showed a decreased mitochondrial membrane potential, NADH level, and oxidative turnover. Oxidative turnover was not sensitive to antimycin A treatment, which suggests that H2-CMXRos oxidation in these cells may be due to lipid peroxidation. In addition, frankly apoptotic cells showed lower cardiolipin levels (by nonyl-acridine orange staining). The efficiency of energy transfer between nonyl-acridine orange and CMXRos was slightly lower in camptothecin-treated nonapoptotic cells and reduced to zero in frankly apoptotic cells.

CONCLUSIONS:

We conclude that, in an initial phase of camptothecin-induced apoptosis, mitochondrial activity is increased and a subtle loss of structural integrity of the mitochondrial membranes takes place. In frankly apoptotic cells, all measured parameters of mitochondrial collapse and lipid peroxidation occurs.

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