Sieprath Tom, Corne Tobias D J, Willems Peter H G M, Koopman Werner J H, De Vos Winnok H
Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
Adv Anat Embryol Cell Biol. 2016;219:149-77. doi: 10.1007/978-3-319-28549-8_6.
Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.
氧化应激源于活性氧（ROS）的产生与细胞抗氧化系统对其清除之间的失衡。特别是在病理条件下，线粒体是细胞ROS的一个相关来源。这些细胞器含有电子传递链，使电子靠近分子氧。尽管仍缺乏全面的了解，但细胞内ROS的产生和线粒体功能也与线粒体形态的变化有关。为了研究活细胞中控制细胞氧化还原平衡的不同因素之间的复杂关系，我们开发了一种基于高内涵显微镜的策略，用于同时定量细胞内ROS水平和线粒体形态功能。在这里，我们总结了细胞内ROS产生和清除的原理，并解释了对活细胞中的ROS和线粒体形态功能进行定量显微镜分析的主要注意事项。接下来，我们描述我们的工作流程，最后，我们说明多参数读数能够明确地对化学扰动细胞以及层粘连蛋白病患者细胞进行分类。
