Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death.
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.
Mattiasson
Gustav
G
Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University, Lund, Sweden. gustav.mattiasson@expbr.lu.se
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism
D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D002118:Calcium