Mitophagy is a natural phenomenon and entails the lysosomal degradation of mitochondria by the autophagy pathway. In recent years, the development of fluorescent pH-sensitive mitochondrial reporters has greatly facilitated the monitoring of mitophagy by distinguishing between cytosolic mitochondria or those delivered to acidic lysosomes. We recently published the mito-QC reporter, which consists of a mitochondrial outer membrane-localised tandem mCherry-GFP tag. This allows the quantification of mitophagy via the increase in red-only mCherry signal that arises when the GFP signal is quenched upon mitochondrial delivery to lysosomes. Here we develop a macro for FIJI, the mito-QC Counter, and describe its use to allow reliable and consistent semi-automated quantification of mitophagy. In this methods article we describe step-by-step how to detect and quantify mitophagy and show that mitophagy levels can be reliably calculated in different cell lines and under distinct stimuli. Finally, we show that the mito-QC Counter can be used to quantify mitophagy in tissues of mito-QC transgenic mice. We demonstrate that mitophagy levels in skeletal muscle correlates with glycolytic activity. Our present data show that the mito-QC Counter macro for FIJI enables the robust quantification of mitophagy both in vitro and in vivo.

中文翻译：

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细胞和组织中线粒体自噬的半自动定量。

线粒体自噬是一种自然现象，需要通过自噬途径对线粒体进行溶酶体降解。近年来，荧光 pH 敏感线粒体报告基因的发展通过区分胞质线粒体或递送至酸性溶酶体的线粒体，极大地促进了线粒体自噬的监测。我们最近发布了 mito-QC 报告基因，它由线粒体外膜定位的串联 mCherry-GFP 标签组成。这允许通过仅红色 mCherry 信号的增加来量化线粒体自噬，当 GFP 信号在线粒体递送至溶酶体时被淬灭时，会产生该信号。在这里，我们为 FIJI 开发了一个宏，即 mito-QC 计数器，并描述了它的用途，以实现可靠且一致的线粒体自噬半自动定量。在这篇方法文章中，我们逐步描述了如何检测和量化线粒体自噬，并表明可以在不同细胞系和不同刺激下可靠地计算线粒体自噬水平。最后，我们证明 mito-QC 计数器可用于量化 mito-QC 转基因小鼠组织中的线粒体自噬。我们证明骨骼肌中的线粒体自噬水平与糖酵解活性相关。我们目前的数据表明，FIJI 的 mito-QC Counter 宏能够对体外和体内线粒体自噬进行稳健的定量。