Pham N A, Robinson B H, Hedley D W
Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada.
Cytometry. 2000 Dec 1;41(4):245-51. doi: 10.1002/1097-0320(20001201)41:4<245::aid-cyto2>3.0.co;2-#.
Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin.
Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy.
Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy.
This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.
由于呼吸链活性缺陷导致线粒体活性氧中间体（ROI）生成增加，这与诸如细胞凋亡等生理过程、线粒体疾病的发病机制以及正常衰老过程的一部分有关。已有的针对呼吸链复合体活性的方法仅限于对单个参数的整体检测。本研究描述了一种基于流式细胞术的方法及其在检测经洋地黄皂苷通透处理的单细胞中呼吸链功能方面的验证。
使用流式细胞术在不同呼吸条件下测量线粒体膜电位（ΔΨm）和活性氧生成。这是通过向经洋地黄皂苷通透处理的细胞中添加底物和抑制剂来实现的。该方法通过测量氧气消耗和ATP生成以及共聚焦显微镜进行了验证。
通过ΔΨm评估的呼吸链复合体活性对底物和抑制剂的反应与通过氧气消耗和ATP合成评估所预测的一致。此外，流式细胞术方法允许同时评估线粒体ROI的生成。这通过共聚焦显微镜观察到的ROI探针羧基-DCF与线粒体探针定位于同一部位得到了证实。
该方法允许在单细胞水平研究呼吸链复合体的功能完整性，从而解决呼吸链复合体功能紊乱与线粒体ROI生成之间的关系。
