Measuring Calcium and ROS by Genetically Encoded Protein Sensors and Fluorescent Dyes

Oxidative modifications of cellular building blocks such as proteins, lipids, and DNA have a major impact on cell behavior, fate, and clinical outcome. Reactive oxygen species (ROS) are important factors that influence these redox processes. Calcium ion (Ca2+) dynamics and signals are also essential regulators of key cellular processes. Therefore, the combined and precise monitoring of ROS and Ca2+ in single cells, with a high spatial and temporal resolution and in physiological environments, is essential to better understand their functional impact. Here, we describe protocols to detect one of the most prominent ROS (hydrogen peroxide, H2O2) using genetically encoded protein sensors and fluorescent dyes. We also provide guidelines on how to simultaneously detect Ca2+ and H2O2 and how to examine the influence of Ca2+ signals on cellular ROS production and vice versa.