A fluorescence imaging based-assay to monitor mitophagy in cultured hepatocytes and mouse liver
10.1016/j.livres.2020.12.002
- Author:
Xiaowen Ma
;
Wen-Xing Ding
- Publication Type:Journal Article
- From:
Liver Research
2021;5(1):16-20
- CountryChina
- Language:English
-
Abstract:
Background and aim
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.
Methods
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.
Results
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.
Conclusions
Cox8-EGFP-mCherry assay can specifically target to mitochondria and be used to monitor mitophagy in vitro and in vivo.
