Ma Xiaowen, Ding Wen-Xing
Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS, USA.
Liver Res. 2021 Mar;5(1):16-20. doi: 10.1016/j.livres.2020.12.002. Epub 2021 Jan 28.
Mitophagy is a lysosomal degradation pathway that selectively removes damaged, aged and dysfunctional mitochondria. Recent advances in understanding mitophagy highlight its importance in various physiological and pathological conditions including liver diseases. However, reliable quantitative assays to monitor mitophagy in cultured cells and in tissues are still scarce.
We describe a detailed protocol for monitoring mitophagy in primary cultured hepatocytes and mouse livers using cytochrome C oxidase subunit 8 (Cox8)-enhanced green fluorescent protein (EGFP)-mCherry, a dual color fluorescence based-imaging method.
Mitochondria are visualized in yellow fluorescence due to the merged EGFP and mCherry signal. In contrast, autolysosome enclosed mitochondria are shown as red puncta due to quenching of EGFP green fluorescence in acidic compartments. Quantifying the number of red-only puncta in each cell can obtain a quantitative measure for mitophagy.
Cox8-EGFP-mCherry assay can specifically target to mitochondria and be used to monitor mitophagy and .
线粒体自噬是一种溶酶体降解途径，可选择性清除受损、老化及功能失调的线粒体。对线粒体自噬认识的最新进展凸显了其在包括肝脏疾病在内的各种生理和病理状况中的重要性。然而，用于监测培养细胞和组织中线粒体自噬的可靠定量检测方法仍然匮乏。
我们描述了一种详细方案，用于使用细胞色素C氧化酶亚基8（Cox8）-增强型绿色荧光蛋白（EGFP）-mCherry（一种基于双色荧光的成像方法）监测原代培养肝细胞和小鼠肝脏中的线粒体自噬。
由于EGFP和mCherry信号合并，线粒体呈现黄色荧光。相比之下，由于酸性区室中EGFP绿色荧光淬灭，被自溶酶体包裹的线粒体呈现为红色斑点。通过量化每个细胞中仅红色斑点的数量可获得线粒体自噬的定量指标。
Cox8-EGFP-mCherry检测可特异性靶向线粒体，用于监测线粒体自噬。
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