Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2 ',7 '-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2 ',7 '-dichlorodihydrofluorescein diacetate, and dihydrorhodamine123

To detect intracellular oxidant formation during reoxygenation of anoxic en dothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydroflu orescein diacetate, dihydrorhodamine 123, or (5 and 6)-carboxy-2/,7'-dichlo rodihydrofluorescein diacetate were added to human umbilical vein endotheli al cells during reoxygenation. None of these fluorescent probes were able t o differentiate the controls from the reoxygenated cells in the confocal mi croscope. However, dihydrofluorescein diacetate demonstrated fluorescence o f linear structures, consistent with mitochondria, in reoxygenated endothel ium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihy drofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish perox idase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and c ompared with the three other popular probes. Though oxidized dihydrofluores cein has higher molar fluorescence, comparison of the reactions of dihydrof luorescein with these other three probes in a cell-free system indicates th at dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very compl ex. Based on the results reported here, it is no longer appropriate to thin k of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions th at may be increased during intracellular oxidant stress. Cell-loading studi es indicate that dihydrofluorescein achieves higher intracellular concentra tions than the second brightest intracellular probe, 2',7'-dichlorodihydrof luorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein di acetate may be a superior fluorescent probe for many cell-based studies. (C ) 1999 Elsevier Science Inc.