We thank Laurie Destroismaisons and Sarah Peyrard for outstanding technical support and Dr.Alexandre Prat for access to the flow cytometer. We would also like to acknowledge Dr. Timothy Miller for his contribution regarding the removal of myelin from the preparations. This work was supported by the Canadian Institutes of Health Research (CIHR) Neuromuscular Research Partnership, Canadian Foundation for Innovation, ALS Society of Canada, the Frick Foundation for ALS Research, CHUM Foundation and Fonds de la Recherche en Sant&#233; du Qu&#233;bec (C.V.V.). Both C.V.V. and N.A. are Research Scholars of the Fonds de la Recherche en Sant&#233; du Qu&#233;bec and CIHR New Investigators. S.P. is supported by the Tim No&#235;l Studentship from the ALS Society of Canada.

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&#8217;Universit&#233; de Montr&#233;al (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.
<img alt="Table 1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> 
Table 1. Buffer Compositions.
1. Collection of Rat Spinal Cord
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Insert a 10 ml syringe, with a 200 &#181;l pipette tip (attached via melting slightly over flame), filled with Phosphate Buffered Saline (PBS), into the vertebral column.</li>
	<li>Flush out the spinal cord by applying a medium amount of pressure to the plunger.</li>
	<li>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.</li>
</ol>
2. Isolation of Spinal Cord Mitochondria (Adapted from Vande Velde et al. 4)
<ol>
	<li>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 &#176;C in a benchtop microcentrifuge.</li>
	<li>Recover supernatant and place in a 5 ml ultracentrifuge tube. Add 750 &#181;l (~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.</li>
	<li>Centrifuge pooled S1 using an ultracentrifuge equipped with a swinging bucket rotor and centrifuge at 17,000 x g for 15 min at 4 &#176;C.</li>
	<li>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 &#176;C in a swinging bucket rotor. Discard the supernatant and gently resuspend the pellet (P3) in 800 &#181;l HB. 
	NOTE: Mitochondria are washed with HB+50 mM KCl to remove any non-specific mitochondrially-associated contaminants.</li>
	<li>In a new 5 ml ultracentrifuge tube, add exactly 800 &#181;l of resuspended pellet (P3).</li>
	<li>To this tube add, 200 &#181;l 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 &#176;C.</li>
	<li>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.</li>
	<li>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 &#176;C. Discard the supernatant and resuspend the pellet in 4 ml HB. Repeat the centrifugation and remove the supernatant.</li>
	<li>Resuspend the final pellet (P7) in 100-200 &#181;l HB and transfer to a 1.7 ml microcentrifuge tube. This sample contains isolated mitochondria.</li>
	<li>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.</li>
</ol>
3. Immunolabeling of Isolated Mitochondria for Flow Cytometry
<ol>
	<li>For each staining mix to be tested, pipette 25 &#181;g 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.</li>
	<li>Centrifuge at 17,000 x g for 2 min at 4 &#176;C in a bench-top microcentrifuge.</li>
	<li>Remove supernatant and resuspend isolated mitochondria in 50 &#181;l Mitochondria Buffer (M Buffer) supplemented with 10% fatty-acid free BSA for 15 min at 4 &#176;C (blocking step). 
	NOTE: During labeling, perform incubations in a refrigerator at 4 &#176;C.</li>
	<li>Add primary antibody (rabbit anti-Mfn2, 20 &#181;g per ml) to tube and incubate for 30 min at 4 &#176;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.</li>
	<li>Wash out unbound primary antibody: Centrifuge at 17,000 x g for 2 min at 4 &#176;C. Remove the supernatant and gently resuspend the pellet in 200 &#181;l M Buffer. Centrifuge at 17,000 x g for 2 min at 4 &#176;C. Remove supernatant and resuspend the pellet in 50 &#181;l M Buffer.</li>
	<li>Add secondary antibody (Donkey anti-rabbit IgG Phycoerythrin (PE), 0.5 &#181;g per ml) to tube and incubate samples for 30 min at 4 &#176;C, protected from light.</li>
	<li>Wash out unbound secondary antibody: Centrifuge at 17,000 x g for 2 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 200 &#181;l M Buffer. Centrifuge at 17,000 x g for 2 min at 4 &#176;C. Remove supernatant and resuspend the pellet in 500 &#181;l M Buffer.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Assaying Mitochondrial Transmembrane Potential and Mitochondrial Superoxide Production by Flow Cytometry
<ol>
	<li>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.
	<ol>
		<li>As a control for TMRM staining, stain isolated mitochondria with 100 nM TMRM in the presence of 100 &#181;M 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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>As a control for mitochondrial superoxide production, stain isolated mitochondria with dye in the presence of 10 &#181;M Antimycin A, an inhibitor of complex III of the respiratory chain that will augment mitochondrial superoxide production.</li>
	</ol>
	</li>
</ol>
5. Acquisition and Analysis of Immunolabeled Isolated Mitochondria by Flow Cytometry
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Determine background labeling from isotype control. Using the isotype control sample, determine the percentage of the mitochondrial population labeling positive for Mfn2 antibody.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+intracellular+organelle+function+using+fluorescent+probes:+from+animalcules+to+quantum+dots.">Examining intracellular organelle function using fluorescent probes: from animalcules to quantum dots.</a> Circ Res. 95, 239-252 (2004).</li><li>Vande Velde, C., Miller, T., Cashman, N. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+association+of+misfolded+ALS-linked+mutant+SOD1+with+the+cytoplasmic+face+of+mitochondria.">Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria.</a> Proc Natl Acad Sci U S A. 105, 4022-4027 (2008).</li><li>Loew, L. M., Tuft, R. A., Carrington, W., Fay, F. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+determination+of+superoxide+release+at+both+sides+of+the+mitochondrial+inner+membrane+by+capillary+electrophoretic+analysis+of+the+oxidation+products+of+triphenylphosphonium+hydroethidine.">Qualitative determination of superoxide release at both sides of the mitochondrial inner membrane by capillary electrophoretic analysis of the oxidation products of triphenylphosphonium hydroethidine.</a> Free Radic Biol Med. 46, 905-913 (2009).</li><li>Otera, H., Ishihara, N., Mihara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+function+and+regulation+of+mitochondrial+fission.">New insights into the function and regulation of mitochondrial fission.</a> Biochim Biophys Acta. 1833, 1256-1268 (2013).</li><li>de Brito, O. 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The authors have nothing to disclose.

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...

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.

<table border="0" cellpadding="0" cellspacing="0" width="1276">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:349px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:543px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Rats</td>
			<td style="width:349px;">Charles River</td>
			<td style="width:119px;">Strain code 400</td>
			<td style="width:543px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dounce homogenizer</td>
			<td style="width:349px;">Kontes Glass Co.&#160;</td>
			<td style="width:119px;">885450-0022</td>
			<td>Duall 22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Microcentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall Legend Micro 17 R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ulara-Clear Ultracentrifuge tubes</td>
			<td style="width:349px;">Beckman Coultier</td>
			<td style="width:119px;">344057</td>
			<td>Transparent, thin walled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sorvall Ultracentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall WX UltraSeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">AH-650 rotor and buckets&#160;&#160;</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">Opti-prep</td>
			<td style="width:349px;">Axis-Shield</td>
			<td style="width:119px;">1114542</td>
			<td>Iodixanol, density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fatty acid free Bovine Serum Albumin</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8806</td>
			<td>Must be fatty acid free for mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sodium succinate dibasic hexahydrate&#160;&#160;</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">S9637</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:267px;">Rabbit anti-Mitofusin2 antibody</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">M6319</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:267px;">Rabbit IgG</td>
			<td style="width:349px;">Jackson Immuno Research&#160;</td>
			<td style="width:119px;">011-000-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Anti-Rabbit IgG PE</td>
			<td style="width:349px;">eBioscience</td>
			<td style="width:119px;">12-4739-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Micro titer tube</td>
			<td style="width:349px;">Bio-Rad</td>
			<td style="width:119px;">223-9391</td>
			<td>For sample acquisition by flow cytometry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoTrackerGreen (MTG)</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M7514</td>
			<td>100 nM: Ex490 nm/Em516 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">TMRM</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">T668</td>
			<td>100 nM: Ex548 nm/Em574 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CCCP</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoSOX Red</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M36008</td>
			<td>5 &#181;M: Ex540 nm/Em600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Antimycin A</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8874</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">LSR II flow cytometer</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BS FACSDiva Software</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FlowJo</td>
			<td style="width:349px;">TreeStar Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Software used for analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">BCA protein assay kit&#160;&#160;</td>
			<td style="width:349px;">Pierce/Thermo Scientific</td>
			<td style="width:119px;">23225</td>
			<td style="width:543px;">Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td>
		</tr>
	</tbody>
</table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

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.

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 ...

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

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.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Research

JoVE Journal

Biology

15.4K Views.  Université de Montréal, CRCHUM. 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.

Video: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...