Measurement of Mitochondrial ROS Formation.

Reactive oxygen species (ROS) play important roles in physiological and pathological processes. Mitochondria, particularly in skeletal and cardiac myocytes, are recognized as the primary site of ROS generation. Detecting oxidative modifications of intracellular or circulating molecules, such as lipids, proteins, and nucleic acids, is commonly employed to indicate ROS formation. However, this approach is indirect and provides limited insights into the spatiotemporal aspects of ROS generation. Understanding these aspects is crucial for comprehending the role of ROS in various pathophysiological conditions. To address this, fluorescent probes can be employed to measure ROS formation, offering a means to investigate ROS generation in both isolated mitochondria and intact cells. This chapter outlines three prominent examples for the use of fluorescent sensors to evaluate mitochondrial ROS formation in either isolated organelles or intact cells. The methods are explained in detail, and an analysis of the limitations of each technique is provided, underscoring potential sources of errors during the assay and the subsequent interpretation of results.