Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Journal of Visualized Experiments : JoVE
Jee-Hyun UmJeanho Yun

Abstract

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.

Citations

Jul 3, 2019·Cancer Discovery·Timothy J HumptonDavid A Tuveson
Mar 25, 2021·Nature Metabolism·Yoon Keun ChoYun-Hee Lee
May 1, 2021·International Journal of Molecular Sciences·Laura DobladoMaria Monsalve
Jul 24, 2021·Frontiers in Cellular and Infection Microbiology·Soo-Jin OhOk Sarah Shin

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