Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.
Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.
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DOI：
10.1016/j.freeradbiomed.2010.01.028
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发表时间：
2010-04-15
影响因子：
7.4
通讯作者：
Kalyanaraman, B.
中科院分区：
文献类型：
--
作者：
Zielonka, Jacek;Kalyanaraman, B.
关键词：
氢乙啶(或二氢乙二胺)(HE)是目前最常用的检测细胞内超氧阴离子的荧光探针。超氧化物与HE反应生成高度特异的红色荧光产物2-羟乙基(2-OH-E+)。在生物体系中，另一种红色荧光产物乙锭(E+)也会生成，其浓度通常比2-羟基-E+高得多。本文综述了细胞和生物系统中超氧化物专一性产物(2-OH-E+)的选择性检测方法及其影响因素。这篇综述中最重要的结论是，使用共聚焦显微镜或其他基于荧光的显微镜方法几乎不可能评估细胞内超氧化物特定产物2-OH-E+的水平，为了全面了解红色荧光的来源，有必要用高效液相色谱法测量细胞内HE和HE的其他氧化产物。线粒体靶向氢乙啶(Mito-HE，MitoSOX Red®)与超氧化物的化学反应类似于HE与超氧化物的反应，因此，所有归因于HE检测的限制也适用于Mito-HE(或Mito-Sox)。
Hydroethidine (or dihydroethidium) (HE) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E+). In biological systems, another red fluorescent product, ethidium (E+), is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we have reviewed the methods to selectively detect the superoxide-specific product (2-OH-E+) and the factors affecting its levels in cellular and biological systems. The most important conclusion of the present review is that it is nearly impossible to assess the intracellular levels of the superoxide specific product, 2-OH-E+, using the confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E+, in order to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red ®) with superoxide is similar to the reactivity of HE with superoxide and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or Mito-SOX) as well.
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DOI：
10.1152/ajpheart.00770.2002
发表时间：
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影响因子：
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作者：
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