Sieprath Tom, Corne Tobias, Robijns Joke, Koopman Werner J H, De Vos Winnok H
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp; Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp.
J Vis Exp. 2017 May 14(123):55449. doi: 10.3791/55449.
Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (ΔΨm and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.
活性氧（ROS）调节包括基因表达、迁移、分化和增殖在内的重要细胞过程。然而，过量的ROS水平会诱导氧化应激状态，伴随对DNA、脂质和蛋白质的不可逆氧化损伤。因此，ROS的定量为细胞健康状况提供了直接指标。由于线粒体是ROS的主要细胞来源和靶点之一，在同一细胞中联合分析线粒体功能和ROS产生对于更好地理解病理生理条件下的相互联系至关重要。因此，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、线粒体膜电位（ΔΨm）和线粒体形态。它基于自动宽场荧光显微镜和对生长在多孔板中的贴壁活细胞的图像分析，并用细胞可渗透的荧光报告分子CM-H2DCFDA（ROS）和TMRM（ΔΨm和线粒体形态）进行染色。与荧光测定法或流式细胞术不同，该策略允许在实验刺激前后以高时空分辨率在单个细胞水平定量亚细胞参数。重要地，该方法基于图像的性质允许除信号强度外还提取形态参数。组合的特征集用于探索性和统计多变量数据分析，以检测亚群、细胞类型和/或处理之间的差异。这里提供了该测定法的详细描述，以及一个示例实验，证明了其在化学扰动后明确区分细胞状态的潜力。
