Quantification of Mitochondrial Reactive Oxygen Species in Living Cells by Using Multi-Laser Polychromatic Flow Cytometry
Quantification of Mitochondrial Reactive Oxygen Species in Living Cells by Using Multi-Laser Polychromatic Flow Cytometry
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DOI：
10.1002/cyto.a.22936
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发表时间：
2016-12-01
期刊：
影响因子：
3.7
通讯作者：
Cossarizza, Andrea
中科院分区：
文献类型：
--
作者：
De Biasi, Sara;Gibellini, Lara;Cossarizza, Andrea
活性氧（ROS）是细胞中不断产生的，主要是由线粒体，作为有氧呼吸的结果。大多数活性氧来自超氧化物，超氧化物迅速转化为过氧化氢。ROS参与调节几种生理和病理过程，并且当细胞的氧化还原状态被实验/生物条件改变时，需要同时测量它们的可能性。流式细胞术是以高通量方式在单细胞水平产生多种信息的主要技术，并以高灵敏度和重现性快速定量测量不同的ROS。在这里，我们描述了一种新的方法来同时检测线粒体过氧化氢和线粒体超氧化物在活细胞中。通过使用荧光染料MitoSOX Red线粒体超氧化物指示剂、线粒体过氧黄1、膜联蛋白-V太平洋蓝缀合物、TO-PRO-3碘化物、抗-CD 4-APC-Cy 7和-CD 8-太平洋橙子mAb进行染色。我们使用这种方法来量化受唐氏综合征影响的患者和年龄和性别匹配的健康供体的CD 4+和CD 8 + T细胞中的线粒体ROS。(C)2016年国际细胞计数促进学会
Reactive oxygen species (ROS) are constantly produced in cells, mainly by mitochondria, as a consequence of aerobic respiration. Most ROS derive from superoxide, which is rapidly converted to hydrogen peroxide. ROS are involved in the regulation of several physiological and pathological processes, and the possibility to measure them simultaneously is needed, when the redox status of the cells is modified by experimental/biological conditions. Flow cytometry is the main technology that generates multiple information at the single cell level in a high-throughput manner, and gives rapid and quantitative measurements of different ROS with high sensitivity and reproducibility. Here, we describe a novel approach to detect simultaneously mitochondrial hydrogen peroxide and mitochondrial superoxide in living cells. The staining has been performed by using the fluorescent dyes MitoSOX Red Mitochondrial Superoxide Indicator, Mitochondria Peroxy Yellow 1, Annexin-V Pacific Blue conjugate, TO-PRO-3 iodide, anti-CD4-APC-Cy7 and -CD8-Pacific Orange mAbs. We used this approach to quantify mitochondrial ROS in CD4+ and CD8+ T cells form patients affected by Down syndrome and age-and sex-matched healthy donors. (C) 2016 International Society for Advancement of Cytometry