Guardrails against Common Pitfalls in Reactive Oxygen Species (ROS) Research: A Chemical Perspective

Abstract Studying the roles of reactive oxygen species (ROS) is at the core of redox biology, with reaction-based probes as one key set of tools. Remarkable progress has been made in our understanding of the roles of various forms of ROS in a wide range of pathophysiological processes. However, there is also an increasing level of recognition of the complex web of interfering factors that could fundamentally affect the rigor of experimental design and results interpretation. The recent discovery of widespread interference posed by solvents (e.g., DMSO), organic buffers (e.g., HEPES, Tris, and MES), cell culture media, and mismatched kinetics between ROS stability and probe reactivity has taken this issue to an elevated level. There is a need for all future ROS research designs to take into consideration these factors for improved rigor. Herein, we aim to critically analyze all these issues by anchoring our discussions on recent experimental findings. We use a combination of two approaches: describing intuitive case studies and building a practical and stepwise workflow to aid application scientists in setting up guardrails against pitfalls due to these interfering factors. Very importantly, we discuss these issues in the chemistry context of probe development, targeted drug delivery, and ROS detection in a biologically relevant fashion. What is not covered are enzyme-based detection methods and a detailed examination of probe selectivity.