Experimental workflows for the accurate identification of mitochondrial redox events

The study of redox biology has been growing constantly since the last decades. Over these years, redox processes have been linked to an extraordinarily wide range of physiological and pathological events, becoming recognized as central mechanisms underlying many of them. In this context, it becomes essential to understand the advantages and limitations of the tools under use, to recognize the specific controls required for each measurement and to accurately distinguish between distinct redox mechanisms. So far, multiple and excellent reviews have dealt with either the tools, the protocols or the mechanisms involved in reactive oxygen species (ROS) production and quenching, a.k.a. redox events. However, a review outlining the workflows to appropriately detect them is still lacking. We define workflow as the combination of tools, methods and mechanistic knowledge that allow the definition of a specific redox event. In this review, we aim to provide an optimal workflow for the research on mitochondrial redox events. To this end, we first summarize the molecular tools available to measure and quench ROS. We then explain the mechanisms of ROS production and scavenging in several of the cellular compartments, with special focus on mitochondria, as well as their implication in physiology and disease. Finally, we use the knowledge in all sections to build a recommended experimental workflow, illustrated by several cases of study. This review will enable the reader to understand how specific mitochondrial redox events can be accurately measured, considering all technical, methodological and mechanistical variables and limitations required for their reliable detection and interpretation.