Background: Mitochondria are key players in many forms of cell death, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes, and release of apoptogenic proteins are involved in these processes. Flow cytometric analysis of isolated mitochondria enables parallel analysis of mitochondrial structure and function in individual mitochondria, and small mitochondrial samples are sufficient for analysis. This article describes a well-characterized protocol for flow cytometric analysis of isolated liver mitochondria that can be used to detect mitochondrial alterations relevant to cell death.
Methods: Fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), and to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide), as well as production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). Calcium-induced mitochondrial swelling was detected as a decrease in SSC. To ensure optimal concentrations of all probes, the effect on mitochondrial respiration was evaluated.
Results: This protocol can be used to determine the purity of the mitochondrial preparation, to detect calcium-induced morphological changes, small mitochondrial de- and hyperpolarizations, as well as physiological changes in ROS generation.
Conclusions: Flow cytometry is a very useful tool to simultaneously analyze several mitochondrial parameters that are important in the induction of mitochondria-mediated cell death.