Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Summary

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Abstract

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Introduction

Mitochondria are the powerhouses of nearly all eukaryotic cells^1,2. In addition to ATP production through oxidative phosphorylation, mitochondria play a vital role in other processes such as bioenergetics, calcium homeostasis, free radical generation, apoptosis, and cellular homeostasis^3,4,5. As mitochondria generate reactive oxygen species (ROS) from multiple complexes in the electron transport chain, they are constantly stimulated by potential oxidative stress, which can eventually lead to structural damage and dysf....

Protocol

1. Cell culture and passaging

NOTE: The protocol is described using routinely cultured mouse embryonic fibroblasts (MEFs) as an example.

1. Culture MEF cells in 10 cm cell culture dishes with 10 mL of Dulbecco's Modified Eagle Medium (DMEM). Incubate at 37 °C and 5% CO₂ and monitor the cells under a microscope at 100x magnification.
2. Perform routine cell passaging.
   1. When the cells reach 80%-90% confluency (every 3 days), wash the cells with 2 mL of Dulbecco's phosphate buffered saline (DPBS). Then add 2 mL of 0.05% trypsin-EDTA for 1 min to dissociate the cells, followed ....

Results

MitoTracker Green is a green-fluorescent mitochondrial stain that is able to accurately localize to mitochondria. The dye can easily stain live cells and is less effective in staining aldehyde-fixed or dead cells (Figure 2). The red-fluorescent lysosome dye LysoTracker Red is capable of labeling and tracking acidic lysosomal organelles and can only stain live cells (Figure 2). Confocal microscope imaging allows the visualization of mitochondria and lysosomes sta.......

Discussion

The protocol described here provides a method for evaluating and monitoring the dynamic process of mitophagy in living cells, involving autophagosomes, lysosomes, and mitochondrial fission, through co-staining with cell-permeant mitochondria and lysosome dyes. The method can also be used to identify mitochondria and assess mitochondrial morphology. Both dyes used in this study should be protected from light, multiple freeze-thaw cycles should be avoided, and the dyes should be stored in single-use aliquots as much as pos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated cell counter	Countstar	IC1000
Cell counting chamber slides	Countstar	12-0005-50
Dulbecco's modified Eagle medium (DMEM)	Corning	10-013-CV
Dulbecco's phosphate-buffered saline (DPBS)	Corning	21-031-CVC
Glass bottom cell culture dish (confocal dish)	NEST	801002
Image J (Rasband, NIH)	NIH		https://imagej.nih.gov/ij/download.html
Krebs–Henseleit(KHB) buffer			Self-prepared
LysoTracker Red	Invitrogen	1818430	100 µmol/L, red-fluorescent lysosome dye
MitoTracker Green	Invitrogen	1842298	200 µmol/L stock, green-fluorescent mitochondria dye
Mouse Embryonic Fibroblasts			Self-prepared
Objective (63x oil lens)	ZEISS	ZEISS LSM 880
Trypsin-EDTA 0.25%	Gibico	Cat# 25200056
ZEISS LSM 880 Confocal Laser Scanning Microscope	ZEISS	ZEISS LSM 880
ZEN Microscopy Software 2.1 (confocal microscope imaging software)	ZEISS	ZEN 2.1