Koopman Werner J H, Verkaart Sjoerd, van Emst-de Vries Sjenet E, Grefte Sander, Smeitink Jan A M, Willems Peter H G M
Department of Membrane Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Cytometry A. 2006 Dec 1;69(12):1184-92. doi: 10.1002/cyto.a.20348.
Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters.
Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images.
At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts.
We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.
线粒体功能障碍可能导致氧化应激增加以及随之而来的细胞铺展变化。在此，我们描述并验证一种同时定量这两个参数的新方法。
用5 -（及-6）-氯甲基-2'，7'-二氯二氢荧光素（CM-H₂DCF）加载人皮肤成纤维细胞，并通过视频速率共聚焦显微镜和实时图像平均法监测其氧化转化为CM-DCF的过程随时间的变化。在对采集的图像进行二值化处理后确定细胞大小。
在最低实际激光输出功率下，CM-DCF的形成呈现零级动力学，表明[CM-H₂DCF]不是限速因素，且[CM-DCF]的形成速率（V(CM-DCF)）是细胞氧化剂水平的函数。对一名健康对照受试者和一名患有氧化磷酸化系统首个复合物NADH：泛醌氧化还原酶缺乏症患者的成纤维细胞进行分析，发现后者细胞中的细胞氧化剂水平显著升高，然而，这并未伴随着细胞铺展的变化。相反，用维生素E的衍生物6-羟基-2,5,7,8-四甲基色满-2-羧酸（生育酚）进行慢性处理，显著降低了对照和成纤维细胞患者细胞中的氧化剂水平和细胞铺展。
我们提出了一种可靠的方法，用于同时定量活细胞中的氧化剂水平和细胞铺展。
