Innovative methods for isolating highly purified mitochondria essential for biomedical studies

Abstract

Mitochondria, being multifunctional and highly complex organelles, possess unique structures and exhibit heterogeneity. In recent decades, the isolation and purification of functional mitochondria have been instrumental for mitochondrial research. As mitochondrial research, including omics, advances, there is a growing demand for the isolation of highly purified mitochondria or individual mitochondria. This paper provides a comprehensive overview of the evolution of mitochondrial purification methods and introduces two innovative and improved techniques for isolating mitochondria from mouse cerebral cortex and in vitro cultured cells. The first method utilizes self-prepared magnetic beads conjugated with anti-TOMM20 antibody for the immunoisolation of highly purified intact mitochondria. The second method utilizes flow cytometry to isolate single mitochondria based on fluorescent protein labeling, allowing for the isolation of mitochondria from a highly heterogeneous population. We provide detailed protocols that aim to benefit the rapidly growing mitochondria research community in assessing mitochondrial function, especially at the single-organelle level.