Reactive Oxygen Species Detection with Fluorescent Probes: Limitations and Recommendations beyond DCFH-DA

Reactive oxygen species (ROS) are highly reactive molecules derived from molecular oxygen that play critical roles in cellular signaling, homeostasis, and the regulation of various physiological and pathological processes. Accurate detection and quantification of ROS are essential for elucidating their roles in health and disease. Despite significant advances in ROS probe development, challenges persist due to their short lifetimes, high reactivity, and their chemical diversity. This perspective article provides a critical evaluation of the limitations of the most commonly applied probe molecule DCFH-DA. Capitalizing on this, recommendations with practical applications in the lab for the specific detection of hydrogen peroxide, hydroxyl radical, singlet oxygen, superoxide anion, and peroxynitrite are provided. By integrating current knowledge on ROS probe technologies, this work aims to guide researchers in reliably assessing ROS in complex biological systems, thereby facilitating a deeper understanding of ROS-mediated processes and their implications for disease research and therapeutic development.