Title: Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry.
Authors: Poot, M; Pierce, R H
Published In Cytometry, (1999 Apr 1)
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.
PubMed ID: 10213196
MeSH Terms: Apoptosis*; Cell Line, Transformed; Electron Transport; Flow Cytometry/methods*; Fluorescent Dyes*; Humans; Intracellular Membranes/metabolism; Lipid Peroxidation; Mitochondria/drug effects; Mitochondria/physiology*; NAD/metabolism; Oxidation-Reduction; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.; Staining and Labeling/methods*