Carboxy-H2DCFDA [5-(and 6)-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate] is a chemically reduced analogue of fluorescein used as an indicator for reactive oxygen species (ROS) in cells. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent Carboxy-H2DCFDA is converted to the highly fluorescent 5-(and 6)-carboxy-2",7"-dichlorofluorescein, with additional negative charges that impede its leakage out of the cell.