Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro

•Fluorometry is among the preferred techniques for monitoring ROS in vitro.•Three frequently used fluorescent probes are DCDHF-DA, DHR 123 and DHE.•To obtain better results, it is crucial to consider modifications to protocols.•Cell culture conditions and characteristics of these probes were considered here.

Reactive oxygen species (ROS) are formed in biological systems by partial reduction of molecular oxygen. The essential role of ROS in maintaining physiological health may be corrupted into oxidative stress by their overproduction or the exhaustion of antioxidant mechanisms. Many studies covering a broad range of methodologies have investigated ROS production and their toxic mechanisms of action. Of these methodologies, fluorometry has been among the preferred techniques. Three frequently used fluorescent probes for in vitro studies are 2′,7′-dichlorodihydrofluorescein diacetate (DCDHF-DA), Dihydrorhodamine 123 (DHR 123) and Dihydroethidium (DHE). Apart from the unavoidable limitations of auto-oxidation, photo-oxidation and photo-conversion, there are also concerns relating to protocol modification for the improved monitoring of ROS. This paper aims to highlight such contributing factors, including cell culture conditions and the characteristics of individual fluorescent probes in the utilization of these selected probes in in vitro systems.