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
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
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DOI：
10.1016/s0891-5849(99)00061-1
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发表时间：
1999-07-01
影响因子：
7.4
通讯作者：
Flaherty, DM
中科院分区：
文献类型：
--
作者：
Hempel, SL;Buettner, GR;Flaherty, DM
为了检测缺氧内皮复氧过程中细胞内氧化剂的形成，在复氧过程中将氧化剂感应荧光探针2',7'-二氯二氢荧光素二乙酸酯、二氢罗丹明123或(5和6)-羧基-2/,7'-二氯二氢荧光素二乙酸酯添加到人脐静脉内皮细胞中。这些荧光探针均无法在共聚焦显微镜中区分对照细胞和复氧细胞。然而，二氢荧光素二乙酸酯在复氧的内皮细胞中表现出与线粒体一致的线性结构的荧光。这项工作测试了这样的假设：二氢荧光素二乙酸盐是检测细胞内氧化剂的更好的荧光探针，因为它对特定的氧化物质更具反应性。为了研究这一点，将二氢荧光素二乙酸酯暴露于各种氧化物质（过氧化氢、超氧化物 [KO2]、过氧亚硝酸盐、一氧化氮、辣根过氧化物酶、三价铁、黄嘌呤氧化酶、细胞色素 c 和脂氧合酶），并与其他三种常用探针进行比较。尽管氧化的二氢荧光素具有较高的摩尔荧光，但在无细胞系统中二氢荧光素与其他三种探针的反应的比较表明，二氢荧光素有时比其他探针的荧光弱。此外，我们发现所有探针的反应性都非常复杂。根据此处报告的结果，不再适合将这些探针视为检测细胞中特定氧化物质（例如 H2O2），而是作为广泛氧化反应的检测器，这些氧化反应在细胞内氧化应激期间可能会增加。细胞加载研究表明，二氢荧光素的细胞内浓度高于第二亮的细胞内探针 2',7'-二氯二氢荧光素。这一事实及其较高的摩尔荧光可能解释了二氢荧光素二乙酸酯的优异亮度。二氢荧光素二乙酸酯对于许多基于细胞的研究来说可能是一种优异的荧光探针。 (C) 1999 爱思唯尔科学公司。
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. (C) 1999 Elsevier Science Inc.