Measurement of mitochondrial ROS formation

Reactive oxygen species (ROS) are involved in both physiological and pathological processes. This widely accepted concept is based more on the effects of antioxidant interventions than on reliable assessments of rates and sites of intracellular ROS formation. This argument applies also to mitochondria that are generally considered the major site for ROS formation, especially in skeletal and cardiac myocytes. Detection of oxidative modifications of intracellular or circulating molecules is frequently used as a marker of ROS formation. However, this approach provides limited information on spatiotemporal aspects of ROS formation that have to be defined in order to elucidate the role of ROS in a given pathophysiological condition. This information can be obtained by means of fluorescent probes that allow monitoring ROS formation in cell-free extracts and isolated cells. Thus, this approach can be used to characterize ROS formation in both isolated mitochondria and mitochondria within intact cells. This chapter describes three major examples of the use of fluorescent probes for monitoring mitochondrial ROS formation. Detailed methods description is accompanied by a critical analysis of the limitations of each technique, highlighting the possible sources of errors in performing the assay and results interpretation.