Analysis of mitochondrial generation and release of reactive oxygen species.
BACKGROUND
Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death.


METHODS
Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain.


RESULTS
The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria.


CONCLUSIONS
The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.
Mattiasson
Gustav
G
Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University, Lund, Sweden. gustav.mattiasson@expbr.lu.se
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000032:analysis*; D013367:Submitochondrial Particles / Q000378:metabolism
D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species