Firstly, one-electron oxidation of DCFH produces DCF•−, and DCF•− reacts with O2 to produce O2•− and subsequently H2O2, increasing ROS level. Secondly, DCFH is susceptible to photo-oxidation. Light exposure will oxidize DCFH and generates ROS.
This fluorescent probe may react with several ROS including hydrogen peroxide, hydroxyl radicals and peroxynitrite. The cell permeant H2DCFDA passively diffuses into cells and is retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by ROS, the non-fluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) when excited with 485nm.
SO this probe only helps to find out the production of Reactive oxygen species inside the cell via green fluorescence. so it doesn't mean that this probe have cytotoxic effects!!!!