Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123

Abstract

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2′,7′-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. 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, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO₂], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think 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 that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2′,7′-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.

Introduction

The detection of H₂O₂ by 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA), (Fig. 1A), in a cell-free system 1, 2 generated interest in the use of fluorescent probes to detect cell-derived oxidants. Shortly after publication of Keston and Brandt’s papers 1, 2, DCHF-DA was used as a detector of intracellular oxidants [3]. In these early studies, the acetate groups were hydrolyzed with base before adding to the cells [3]. Fluorescence was then detected in cell lysates [3]. Cell biologists did not use DCHF-DA to detect intracellular oxidants in live cells until 1983 when Bass demonstrated that DCHF-DA could be used with flow cytometry to detect oxidant formation by activated neutrophils [4]. Bass speculated that the probe diffuses into the cell, intracellular esterases hydrolyze the acetate groups, and the resulting 2′,7′-dichlorofluorescin (DCHF) then reacts with intracellular oxidants resulting in the observed fluorescence [4]. Since then, there have been numerous reports using DCHF-DA to measure intracellular oxidants. Much of this work has used flow cytometry 4, 5, 6 and cells capable of a respiratory burst, such as macrophages or polymorphonuclear leukocytes 4, 5, 6, 7. More recently, investigators have used DCHF-DA to study other cells, such as the endothelial cell 8, 9. Additional probes have also been introduced. Dihydrorhodamine 123 (DHR123), (Fig. 1B), is presumed to localize to mitochondria [10], whereas 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (5&6DH-DA), (Fig. 1B), is supposed to be retained in the cell better than DCHF, thereby yielding more fluorescence 11, 12.

Initially, three intracellular fluorescent probes (DCHF-DA, DHR123, and 5&6DH-DA) were used in our laboratory as a tool to detect intracellular oxidants formed during reoxygenation of anoxic endothelial cells 13, 14, 15, 16, 17. We speculated that the mitochondria would be a likely source of oxidants during reoxygenation [17] and attempted to use confocal microscopy with these fluorescent probes to detect mitochondria-derived species during reoxygenation. However, none of these probes gave a fluorescent signal that was significantly different than the room air controls. Subsequently, we found that dihydrofluorescein diacetate (HFLUOR-DA) readily imaged linear intracellular structures having the appearance of mitochondria during reoxygenation of the endothelium. These findings indicate that HFLUOR-DA, first described as fluorescein in 1871, may be a useful tool for other investigators studying intracellular oxidants 18, 19.

The present study tests the hypothesis that HFLUOR-DA is superior for detecting intracellular oxidants by confocal microscopy because it is more reactive toward oxidants than the other three probes. Our results indicate that the superiority of HFLUOR-DA for imaging intracellular oxidants by confocal microscopy is not due simply to enhanced reactivity. We find that the reactivity of all four probes is very complex and each probe has a unique reactivity toward specific oxidizing species. Based on results reported here, it is no longer appropriate to think of these probes as detecting a specific species, such as H₂O₂ 6, 8, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress [20]. One explanation for the increased fluorescence of HFLUOR-DA, compared with the other fluorescent probes, is a higher molar fluorescence. In addition, HFLUOR achieves higher intracellular concentrations than DCHF, thus more compound is available for oxidation to fluorescent species.