Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Summary

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Abstract

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Introduction

Mitochondria are highly dynamic organelles that undergo multiple rounds of fission and fusion, are transported to sites of high energy demand and respond rapidly to physiological stimuli¹. Since it is increasingly recognized that mitochondria within different tissues, even different cellular compartments, have distinct functional profiles, new methods are needed to identify these distinct mitochondrial subsets.

Microscopy provides a means whereby individual mitochondria can be visualized and the presence of a protein at or in mitochondria can be determined by immunofluorescence². However, quantitative analysis by this ....

Protocol

Animals used in this study were treated in strict accordance to a protocol (N08001CVsr) approved by the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM) Institutional Committee for the Protection of Animals which follows national standards as outlined by the Canadian Council on Animal Care (CCAC).

Prepare all reagents required to perform this protocol (Table 1). All other details regarding equipment, supplies and suppliers can be found in List of Materials.

Results

Mitochondria derived from rat spinal cords can be immunolabeled with an antibody targeted to Mitofusin2 (Mfn2), a protein implicated in the fusion of the outer membrane of mitochondria⁸. Following isolation and labeling with a Mfn2 specific antibody and a fluorescently conjugated secondary antibody, mitochondria are processed by flow cytometry (Figure 1). Following data acquisition, samples are analyzed using flow cytometry analysis software, by first visualizing all collected events on a dot .......

Discussion

It is increasingly evident that mitochondria are key players in both normal physiology and disease. While immunoblotting can determine which proteins are found within mitochondria or at the mitochondrial surface in a certain condition, this method reports on the average of the entire population. This method cannot yield information about relative abundances of mitochondrial subpopulations or subsets. While it has been previously assumed that all mitochondria are created equally, the field is increasingly recognizing that.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rats	Charles River	Strain code 400	Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.
Dounce homogenizer	Kontes Glass Co.	885450-0022	Duall 22
Microcentrifuge	Thermo Scientific		Sorvall Legend Micro 17 R
Ultra-Clear Ultracentrifuge tubes	Beckman Coulter	344057	Transparent, thin walled
Sorvall Ultracentrifuge	Thermo Scientific		Sorvall WX UltraSeries
AH-650 rotor and buckets	Thermo Scientific
Opti-prep	Axis-Shield	1114542	Iodixanol, density gradient medium
Fatty acid free Bovine Serum Albumin	Sigma	A8806	Must be fatty acid free for mitochondria
Sodium succinate dibasic hexahydrate	Sigma	S9637
Rabbit anti-Mitofusin2 antibody	Sigma	M6319
Rabbit IgG	Jackson Immuno Research	011-000-003
Anti-Rabbit IgG PE	eBioscience	12-4739-81
Micro titer tube	Bio-Rad	223-9391	For sample acquisition by flow cytometry
MitoTrackerGreen (MTG)	Invitrogen	M7514	100 nM: Ex 490 nm/Em 516 nm
TMRM	Invitrogen	T668	100 nM: Ex 548 nm/Em 574 nm
CCCP	Sigma	C2759
MitoSOX Red	Invitrogen	M36008	5 µM: Ex 540 nm/Em 600 nm
Antimycin A	Sigma	A8874
LSR II flow cytometer	BD
BD FACSDiva Software	BD
FlowJo	TreeStar Inc.		Software used for analysis
BCA protein assay kit	Pierce/Thermo Scientific	23225	Bradford assay is not recomended as it is not compatible with high concentrations of SDS