Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

Summary

In this study, we describe a step-by-step protocol and emphasize the key details for determining morphological characteristics of mitochondria in live cells, including sample preparation, image acquisition, and data analysis. This method is commonly used to examine mitochondrial morphology for studying various conditions.

Abstract

The dynamic balance of mitochondrial fusion and fission directly contributes to mitochondrial homeostasis, which influences numerous cellular functions in addition to adenosine triphosphate (ATP) homeostasis. Therefore, assessing mitochondrial morphology under stress conditions is essential for mechanistic research. This study describes a detailed protocol for analyzing mitochondrial morphology, encompassing the preparation of a MitoTracker solution, staining of mitochondria, optimization of imaging parameters, and detection of morphological features. MitoTrackers are commonly used, cost-effective mitochondrion-specific dyes. However, some changes in mitochondrial morphology may occur owing to inappropriate handling, which can be unperceivable and fail to reflect the true state of mitochondria. Therefore, it is necessary to understand how to analyze changes in mitochondrial morphology using MitoTrackers. The protocol utilized SH-SY5Y cells stimulated with 1-methyl-4-phenylpyridinium iodide (MPP⁺) to illustrate the protocol of mitochondrial morphological analysis. Compared with control cells, MPP⁺-stimulated cells exhibited smaller and more fragmented mitochondria, with morphological parameters indicating decreased mitochondrial footprint. These results suggest that MitoTracker staining is an effective and feasible method for mitochondrial morphological analysis that (with minor modifications) can be applied to study various conditions.

Introduction

Mitochondrial morphology is maintained by the dynamic balance of mitochondrial fusion and fission, and hence, affects energy homeostasis and numerous other cellular functions, leading to various pathologies such as neurodegenerative diseases, cancer, and inflammation^1,2,3,4,5. To observe mitochondrial morphology, several mitochondrion-selective stains, including both probes and antibodies, have been developed for use with a confocal microscope. Some mitochondrion-specific probes, like tetramethylrosamine an....

Protocol

1. Preparation

1. Dissolve MPP⁺ with dimethyl sulfoxide (DMSO) to make a 1 mM stock solution and store it at -30 °C in the dark (see Table of Materials).
2. Dissolve 50 µg of MitoTracker (MitoTracker Red CMXRos) in 94 µL of DMSO to make a 1 mM stock solution. Aliquot the stock solution into several microcentrifuge tubes and store protected from light at -30 °C until further use.
3. Incubate SH-SY5Y cells in flasks with Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified environment with 5% CO₂

Results

This protocol outlines a detailed, step-by-step procedure for mitochondrial morphological analysis through three interdependent optimization modules: standardization of specimen preparation protocols, systematic optimization of optical parameters for confocal microscopy imaging, and computational image processing guidelines. Optimal sample preparation protocol is critical for obtaining reliable mitochondrial imaging data. Systematic evaluation of MitoTracker Red CMXRos concentrations demonstrated concentration-dependent .......

Discussion

The present study establishes a systematic methodology for analyzing mitochondrial morphology in live cells under stress conditions. Crucial procedural steps in mitochondrial imaging involve: an appropriate concentration of MitoTracker probes, thoroughly washing confocal dishes prior to image acquisition, minimizing disturbances to the cellular state, and setting suitable imaging parameters.

Using higher concentrations of MitoTracker can induce cytotoxicity, rendering mitochondria sensitive to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-methyl-4-phenylpyridinium iodide (MPP+)	Macklin	M875357	Used to induce oxidative stress
Confocal dish	NEST	801001	Used to culture the cells
Confocal microscope	Nikon	C2	Used to capture images
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12	ThermoFisher Scientific	11320033	Used to provide nutrition to cells
Dimethyl sulfoxide (DMSO)	Beyotime	ST038	Used to dissolve the mitotracker probe
Fetal bovine serum (FBS)	ThermoFisher Scientific	A3161001C	Used to provide nutrition to cells
ImageJ	National Institutes of Health	ImageJ	Used to analyze mitochondrial morphology
MitoTracker Red CMXRos	ThermoFisher Scientific	M7512	Used to visualize mitochondria
Phosphate Buffer Saline (PBS)	ThermoFisher Scientific	C10010500BT	Used to wash the cells on confocal dishes
SH-SY5Y cell	ATCC	CRL-2266	Used to demonstrate the workflow of analyzing mitochondrial morphology
TI-SH-U Stage Adapter	Nikon	MEC59110	Used to secure specimens