Department of Immunology, University of Toronto, Toronto, ON, Canada.
Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, ON, Canada.
Biotechniques. 2020 Oct;69(4):249-256. doi: 10.2144/btn-2020-0071. Epub 2020 Aug 18.
Mitophagy is the process by which mitochondria are selectively targeted and removed via autophagic machinery to maintain mitochondrial homeostasis in the cell. Recently, flow cytometry-based assays that utilize the fluorescent mtKeima reporter system have allowed for quantitative assessment of mitophagy at a single-cell level. However, clear guidelines for appropriate flow cytometry workflow and downstream analysis are lacking and studies using flow cytometry in mtKeima-expressing cells often display incorrect and arbitrary binary mitophagic or nonmitophagic cutoffs that prevent proper quantitative analyses. In this paper we propose a novel method of mtKeima data analysis that preserves subtle differences present within flow cytometry data in a manner that ensures reproducibility.
线粒体自噬是通过自噬机制选择性靶向和清除线粒体以维持细胞中线粒体动态平衡的过程。最近，基于流式细胞术的测定方法利用荧光 mtKeima 报告系统允许在单细胞水平上对线粒体自噬进行定量评估。然而，目前缺乏适当的流式细胞术工作流程和下游分析的明确指南，并且在使用流式细胞术在表达 mtKeima 的细胞中进行的研究通常显示不正确和任意的二进制线粒体自噬或非线粒体自噬截止值，从而阻止了适当的定量分析。在本文中，我们提出了一种新的 mtKeima 数据分析方法，以确保可重复性的方式保留流式细胞术数据中存在的细微差异。
