Um Jee-Hyun, Kim Young Yeon, Finkel Toren, Yun Jeanho
Peripheral Neuropathy Research Center, College of Medicine, Dong-A University; Department of Biochemistry, College of Medicine, Dong-A University.
Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine.
J Vis Exp. 2018 Aug 12(138):58099. doi: 10.3791/58099.
Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.
线粒体自噬是一种利用自噬机制选择性清除受损或多余线粒体的过程。已发现线粒体自噬缺陷与多种人类疾病密切相关，包括神经退行性疾病、癌症和代谢性疾病。此外，最近的研究表明，线粒体自噬参与正常细胞过程，如分化和发育。然而，线粒体自噬的确切作用和分子机制仍需进一步研究。因此，开发一种强大且便捷的方法来测量线粒体自噬活性的变化至关重要。在此，我们描述了一种详细的方案，通过使用线粒体靶向荧光蛋白Keima（mt-Keima）的流式细胞术定量评估线粒体自噬活性。这种流式细胞术检测方法比传统的基于显微镜或免疫印迹的方法能更快速、灵敏地分析线粒体自噬活性。该方案可应用于分析各种细胞类型中的线粒体自噬活性。
