Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry
✍ Scribed by Martin Poot; Robert H. Pierce
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 93 KB
- Volume
- 35
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
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-(4Ј-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 (H 2 -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 H 2 -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 nonylacridine 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. Cytometry 35:311-317, 1999. 1999 Wiley-Liss, Inc.