Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy
Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy
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DOI：
10.1038/nprot.2016.094
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发表时间：
2016-09-01
期刊：
影响因子：
14.8
通讯作者：
Koopman, Werner J. H.
中科院分区：
文献类型：
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作者：
Iannetti, Eligio F.;Smeitink, Jan A. M.;Koopman, Werner J. H.
线粒体在细胞（病理）生理学中起着核心作用，它们显示出高度可变的形态，这可能与它们的功能状态有关。在这里，我们提出了一个协议，允许无偏和自动量化线粒体的“形态功能”（即，形态和膜电位）、细胞参数（大小、汇合）和核参数（数量、形态）。将细胞在96孔板中培养并用四甲基罗丹明甲酯（TMRM）、钙黄绿素-AM（乙酰氧基-甲酯）和Hoechst 33258染色。接下来，使用自动显微镜采集多光谱荧光图像，并进行处理以提取44个描述符。随后，对描述符数据进行基于主成分分析（PCA）的质量控制（QC）算法，并使用单变量、双变量和多变量分析进行解释。该协议需要的时间投资类似于4 h分布在2 d。虽然它是专门为临床前研究中广泛使用的PHSF开发的，但该方案可移植到其他细胞类型，并且可以扩大规模以用于高内容筛选。
Mitochondria have a central role in cellular (patho) physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), calcein-AM (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are subjected to a quality control (QC) algorithm based upon principal component analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of similar to 4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening.