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 method is labor intensive and is more suitable for experiments using immortalized or primary cell lines. The study of individual mitochondria derived from tissue is significantly more difficult and most methods do not allow for easy identification of mitochondrial subsets concurrently with the evaluation of mitochondrial function³.

In order to address this hurdle, a novel method to immunolabel mitochondria isolated from rodent tissues and subsequently analyzed by flow cytometry has been developed. This allows for the rapid detection and quantification of proteins localized to the mitochondrial outer membrane, which compared to analysis by microscopy, is much less labor intensive and permits the analysis of thousands of mitochondria in a single sample. This assay can be applied to monitor the fate and relative amount of mitochondrial outer membrane proteins that are thought to be constitutively present at the mitochondria, the recruitment of proteins to the mitochondrial surface, or the detection of proteins mislocalized to the mitochondria in pathological conditions. Moreover, the incorporation of conventional fluorescent indicator dyes permits the simultaneous evaluation of certain aspects of mitochondrial function in distinct mitochondrial subpopulations.

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.

Table 1. Buffer Compositions.

1. Collection of Rat Spinal Cord

1. Deeply anaesthetize the rat (Sprague Dawley) with 4% isoflorane. Verify anaesthetization by a lack of reflex upon pinching of the forepaw. Euthanize the rat by decapitation via guillotine. This method of euthanasia is preferred over others, which might distort the spinal cord.
2. Cut the skin of the back to expose the spine. Cut the spinal column with bone scissors just above the pelvic bone. Visualize the opening of the vertebral column.
3. Insert a 10 ml syringe, with a 200 µl pipette tip (attached via melting slightly over flame), filled with Phosphate Buffered Saline (PBS), into the vertebral column.
4. Flush out the spinal cord by applying a medium amount of pressure to the plunger.
5. If any blood is present on the spinal cord, rinse with PBS before proceeding to the next step.
   NOTE: This method has also been validated for brain and liver. If including these tissues, collect half the brain from the euthanized rat and a piece of liver equal in weight to the spinal cord. All other steps remain identical.

2. Isolation of Spinal Cord Mitochondria (Adapted from Vande Velde et al. ⁴)

1. Collect the whole intact spinal cord and place in 5 ml glass homogenizer with 5 volumes (~3.25 ml) Homogenization Buffer (HB). For optimal recovery of isolated mitochondria, perform all steps on ice or in cold room. Homogenize tissue by hand until no large pieces of tissue remain, approximately eight strokes. Place homogenate in two (2 ml) or three (1.7 ml) microcentrifuge tubes. Centrifuge 1,300 x g for 10 min at 4 °C in a benchtop microcentrifuge.
2. Recover supernatant and place in a 5 ml ultracentrifuge tube. Add 750 µl (~0.5 volumes) HB to the pellet-containing microcentrifuge tube and gently resuspend the pellet. Repeat centrifugation and resuspension steps two more times. Pool all supernatants (S1a and S1b) into the same 5 ml ultracentrifuge tube above. This step serves to remove small debris.
3. Centrifuge pooled S1 using an ultracentrifuge equipped with a swinging bucket rotor and centrifuge at 17,000 x g for 15 min at 4 °C.
4. Keep supernatant (S2) for further processing if the cytosolic fraction is of interest (for example for Western blot analysis). Resuspend the pellet (P2), the crude mitochondrial fraction, in 4 ml HB+50 mM KCl. Centrifuge 17,000 x g for 15 min at 4 °C in a swinging bucket rotor. Discard the supernatant and gently resuspend the pellet (P3) in 800 µl HB.
   NOTE: Mitochondria are washed with HB+50 mM KCl to remove any non-specific mitochondrially-associated contaminants.
5. In a new 5 ml ultracentrifuge tube, add exactly 800 µl of resuspended pellet (P3).
6. To this tube add, 200 µl of Iodixanol (density gradient medium), thereby creating a final concentration of 12% Iodixanol. Mix the contents of the tube gently, but thoroughly via pipetting with a P1000. Addition of Iodixanol first, and then resuspended pellet (P3) may be preferable to facilitate thorough mixing. Centrifuge in an ultracentrifuge equipped with a swinging bucket rotor at 17,000 x g for 15 min at 4 °C.
7. NOTE: Liver does not contain myelin and therefore this step is not necessary if only the liver is being processed. However, if liver is being processed concurrently with CNS mitochondria, it is recommended to treat all tissues equally.
8. Aspirate the layer of myelin at the top of the tube and carefully remove and discard the supernatant. Pellet may be loose. Resuspend the pellet in 4 ml HB. Centrifuge again at 17,000 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 4 ml HB. Repeat the centrifugation and remove the supernatant.
9. Resuspend the final pellet (P7) in 100-200 µl HB and transfer to a 1.7 ml microcentrifuge tube. This sample contains isolated mitochondria.
10. Proceed to protein quantification. Dilute samples and the standard curve in 2% sodium dodecyl sulfate (SDS) to ensure adequate solubilization of mitochondria during protein quantification.

3. Immunolabeling of Isolated Mitochondria for Flow Cytometry

1. For each staining mix to be tested, pipette 25 µg of isolated mitochondria into a 1.7 ml microcentrifuge tube. Include an unstained sample in each experiment; and for each antibody, be sure to include a sample for the appropriate isotype control.
2. Centrifuge at 17,000 x g for 2 min at 4 °C in a bench-top microcentrifuge.
3. Remove supernatant and resuspend isolated mitochondria in 50 µl Mitochondria Buffer (M Buffer) supplemented with 10% fatty-acid free BSA for 15 min at 4 °C (blocking step).
   NOTE: During labeling, perform incubations in a refrigerator at 4 °C.
4. Add primary antibody (rabbit anti-Mfn2, 20 µg per ml) to tube and incubate for 30 min at 4 °C.
   NOTE: Determine optimal concentration of each antibody empirically by titration. Due to variability in concentration and/or purity, different lots of the same antibody from the same manufacturer may lead to different results; therefore titration is needed for each new lot of antibody.
5. Wash out unbound primary antibody: Centrifuge at 17,000 x g for 2 min at 4 °C. Remove the supernatant and gently resuspend the pellet in 200 µl M Buffer. Centrifuge at 17,000 x g for 2 min at 4 °C. Remove supernatant and resuspend the pellet in 50 µl M Buffer.
6. Add secondary antibody (Donkey anti-rabbit IgG Phycoerythrin (PE), 0.5 µg per ml) to tube and incubate samples for 30 min at 4 °C, protected from light.
7. Wash out unbound secondary antibody: Centrifuge at 17,000 x g for 2 min at 4 °C. Remove the supernatant and resuspend the pellet in 200 µl M Buffer. Centrifuge at 17,000 x g for 2 min at 4 °C. Remove supernatant and resuspend the pellet in 500 µl M Buffer.
8. To ensure events are in fact mitochondria, stain isolated mitochondria with a mitochondria specific fluorescent dye for 15 min at RT, protected from light. If staining of other functional parameters (mitochondrial transmembrane potential or superoxide production) is desired, proceed to step 4. If not, proceed to step 5 for acquisition.
   NOTE: It is important to verify that the emission spectrum of the secondary antibody is compatible with that of the functional dyes. For example, if verifying mitochondrial purity with a commercial dye with spectral properties similar to FITC and transmembrane potential with Tetramethylrhodamine methyl ester (TMRM), a viable secondary antibody would be allophycocyanin (APC: Ex 650 nm/Em 660 nm). Add compensations controls, i.e., a sample immunolabeled or stained with a single fluorophore, when applicable.
9. Transfer to a tube suitable for loading flow cytometer. (To facilitate the small sample size, a microtiter tube is placed inside 3 ml flow cytometer tube.) Keep samples on ice and proceed immediately to flow cytometer for acquisition.

4. Assaying Mitochondrial Transmembrane Potential and Mitochondrial Superoxide Production by Flow Cytometry

1. Verify that isolated mitochondria have an intact transmembrane potential by staining with 100 nM TMRM (Ex 548 nm/Em 574 nm)⁵, at step 3.8, for 15 min at RT, protected from light. For comparison of transmembrane potential between samples and populations, use of lower/non-quenching concentrations of TMRM (1 to 30 nM), may be more appropriate⁶.
   1. As a control for TMRM staining, stain isolated mitochondria with 100 nM TMRM in the presence of 100 µM carbonyl cyanide m-chloro phenyl hydrazine (CCCP), a mitochondrial uncoupler that will depolarize mitochondria. The concentration of CCCP required to depolarize the mitochondria may be less if lower concentrations of dye are used.
2. Verify that isolated mitochondria produce mitochondrial superoxide by staining with an appropriate mitochondrial superoxide indicator⁷, also at step 3.8, for 15 min at RT, protected from light.
   1. As a control for mitochondrial superoxide production, stain isolated mitochondria with dye in the presence of 10 µM Antimycin A, an inhibitor of complex III of the respiratory chain that will augment mitochondrial superoxide production.

5. Acquisition and Analysis of Immunolabeled Isolated Mitochondria by Flow Cytometry

1. Instrument set up: Switch voltages from linear to log mode to facilitate analysis of isolated mitochondria and set voltages (FSC: 450; SSC: 250). Ensure that events are collected in FSC-A (area) mode as well as FSC-W (width) and FSC-H (height), to be able to exclude doublets (i.e., two events, passing through the detector at the same time) in analysis post-data collection. Set the number of events to be collected to 100,000. Acquire compensation controls, if applicable.
2. Data acquisition: Before data acquisition, avoid vortexing samples. Instead mix by gently tapping tube. Initially collect events at a low pressure, during gating. Gate on total population. Adjust voltages of histograms accordingly, usually the peak of the unstained sample will correspond to the second decade (10²). Once gates are established, and samples are being processed, the pressure can be switched to high.
3. Analysis: Visualize doublets by plotting FSC-W versus FSC. Identify singlets and doublets. Gate on singlets. Select the mitochondrial population by gating on events that are stained positively with a mitochondrial-selective dye.
4. Determine background labeling from isotype control. Using the isotype control sample, determine the percentage of the mitochondrial population labeling positive for Mfn2 antibody.

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