An update on methods and approaches for interrogating mitochondrial reactive oxygen species production

The chief ROS formed by mitochondria are superoxide ([Formula: see text]) and hydrogen peroxide (H(2)O(2)). Superoxide is converted rapidly to H(2)O(2) and therefore the latter is the chief ROS emitted by mitochondria into the cell. Once considered an unavoidable by-product of aerobic respiration, H(2)O(2) is now regarded as a central mitokine used in mitochondrial redox signaling. However, it has been postulated that [Formula: see text] can also serve as a signal in mammalian cells. Progress in understanding the role of mitochondrial H(2)O(2) in signaling is due to significant advances in the development of methods and technologies for its detection. Unfortunately, the development of techniques to selectively measure basal [Formula: see text] changes has been met with more significant hurdles due to its short half-life and the lack of specific probes. The development of sensitive techniques for the selective and real time measure of [Formula: see text] and H(2)O(2) has come on two fronts: development of genetically encoded fluorescent proteins and small molecule reporters. In 2015, I published a detailed comprehensive review on the state of knowledge for mitochondrial ROS production and how it is controlled, which included an in-depth discussion of the up-to-date methods utilized for the detection of both superoxide ([Formula: see text]) and H(2)O(2). In the article, I presented the challenges associated with utilizing these probes and their significance in advancing our collective understanding of ROS signaling. Since then, many other authors in the field of Redox Biology have published articles on the challenges and developments detecting [Formula: see text] and H(2)O(2) in various organisms [[1], [2], [3]]. There has been significant advances in this state of knowledge, including the development of novel genetically encoded fluorescent H(2)O(2) probes, several [Formula: see text] sensors, and the establishment of a toolkit of inhibitors and substrates for the interrogation of mitochondrial H(2)O(2) production and the antioxidant defenses utilized to maintain the cellular H(2)O(2) steady-state. Here, I provide an update on these methods and their implementation in furthering our understanding of how mitochondria serve as cell ROS stabilizing devices for H(2)O(2) signaling.