Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Bilin Liu; Anqi Li; Yuan Qin; Lei Chen; Meng Gao; Guohua Gong

doi:10.3791/64647

Visualización de la mitofagia con tintes fluorescentes para mitocondrias y lisosomas

JoVE Journal
Biology
Author Produced

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Visualización de la mitofagia con tintes fluorescentes para mitocondrias y lisosomas

Full Text

6,254 Views

07:56 min

November 30, 2022

DOI: 10.3791/64647-v

Bilin Liu^1,2, Anqi Li¹, Yuan Qin³, Lei Chen¹, Meng Gao¹, Guohua Gong^1,2

¹Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology,Tongji University, ²Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences,Wenzhou Medical University, ³Department of Pharmacy, Shanghai East Hospital,Tongji University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study investigates mitophagy, the process of mitochondrial quality control, using a live-cell imaging approach. A protocol utilizing green-fluorescent mitochondria dye and red-fluorescent lysosome dye is outlined to observe mitophagy in mouse embryonic fibroblast cells.

Key Study Components

Research Area

Cell biology
Mitophagy and mitochondrial dynamics
Mitochondria-related diseases

Background

Mitophagy plays a crucial role in maintaining mitochondrial homeostasis.
Reliable quantitative assays for mitophagy in vivo are lacking.
This technique may pave the way for new treatments of mitochondria-related diseases.

Methods Used

Live-cell imaging using confocal microscopy
Mouse embryonic fibroblast (MEF) cells
Cell-permeant fluorescent dyes for staining mitochondria and lysosomes

Main Results

Clear imaging revealed colocalization of mitochondria and lysosomes, indicating active mitophagy.
The technique allows quantification of mitophagy by counting yellow co-localized structures.
Demonstrated potential to analyze mitochondrial number and morphology.

Conclusions

This method enhances the ability to study mitophagy in living cells.
It provides insights into the role of mitophagy in various human diseases.

Frequently Asked Questions

What is mitophagy?

Mitophagy is the selective degradation of damaged or surplus mitochondria via autophagy, crucial for mitochondrial quality control.

What is the significance of observing mitophagy?

Studying mitophagy is important for understanding its role in maintaining cellular health and its implications in mitochondrial dysfunction-related diseases.

How are live cells prepared for the mitophagy assay?

Cells are cultured in DMEM, treated with trypsin for dissociation, and resuspended in a diluted cell suspension for imaging.

What dyes are used in this method?

A cell-permeant green-fluorescent dye for mitochondria and a red-fluorescent dye for lysosomes are utilized.

What are the imaging conditions required?

Cells need to be imaged at 37 degrees Celsius using confocal microscopy with specific excitation wavelengths for dual-channel detection.

What can be inferred from the overlap of green and red fluorescence?

The overlap indicates active mitophagy, revealing the interaction between damaged mitochondria and lysosomes.

How does this technique contribute to disease research?

This method can uncover the relationship between mitophagy and various human diseases, facilitating the exploration of new treatments.

La mitofagia es el principal mecanismo de control de calidad mitocondrial. Sin embargo, la evaluación de la mitofagia in vivo se ve obstaculizada por la falta de ensayos cuantitativos fiables. Aquí se presenta un protocolo para la observación de la mitofagia en células vivas utilizando un colorante mitocondrial verde-fluorescente permean celular y un colorante lisosómico rojo-fluorescente.

Este método ayuda a observar la mitofagia en células vivas utilizando un colorante mitocondrial verde-fluorescente perceptible celular y un colorante lisosómico rojo-fluorescente. La mitofagia juega un papel importante en el mantenimiento de la homeostasis de las mitocondrias y varios aspectos de la función celular. Esta técnica podría proporcionar un nuevo enfoque para el tratamiento de enfermedades relacionadas con las mitocondrias.

Este procedimiento no es muy complicado. La captura de imágenes claras es muy importante para contar la superposición de mitocondrias y lisosomas. Para comenzar, cultive fibroblastos embrionarios de ratón o células MEF en una placa de cultivo celular de 10 centímetros con 10 mililitros de Dulbecco's Modified Eagle Medium o DMEM.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Biología Número 189