Dichlorofluorescein is a fluorescent marker commonly used for measuring intracellular reactive oxygen species (ROS). Whereas the use of dichlorofluorescein (DCF) have been frequent, investigation and discussion of the chemical and biological parameters relevant to DCF measurement of ROS has been limited. The discussion of the chemical parameters of DCF presented by Wardman (1) is welcomed, and we agree that the discussed chemical properties of DCF are central for both experimental design and results interpretation.

From our experiments, we would like to add that certain biological parameters are also important to consider in the use of the dichlorofluorescein probe to measure radiation-induced ROS. Specifically with regard to cellular probe loading, leakage and interchange, controlling for these biological parameters is equally important for proper experimental design and data interpretation.

Dichlorofluorescin (DCFH) is loaded into cells in the form of dichlorofluorescin diacetate (DCFH-DA) that once present...

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