This work was supported by the Canadian Institutes of Health Research (148808 and 148792), SE was supported by a Heart and Stroke Foundation, personnel award from the Ontario Provincial Office, March of Dimes, Ted Rogers Centre for Heart Research and the Peter Munk Cardiac Centre. XCC holds a CIHR Banting Fellowship. LA holds a Heart &amp; Stroke/Richard Lewar Studentship Award.

Ethics statement: This protocol has been reviewed and approved by the Animal Care Committee at the University Health Network (Toronto, Canada) and is in compliance with the Canadian Council on Animal Care.
1. Buffer Preparation
<ol>
	<li>Prepare HBB buffer (Hank&#39;s Balanced Salt Solution (HBSS), 2% heat inactivated bovine serum, 0.2% bovine serum albumin). Add 10 mL of heat inactivated bovine serum and 1 g of bovine serum albumin to 500 mL of HBSS. Filter sterilize through a 0.2 &#181;m filter and store at 4 &#176;C.</li>
	<li>Prepare FACS buffer (Phosphate buffered saline (PBS), 2% heat inactivated bovine serum, 1 mM Ethylenediaminetetraacetic acid (EDTA, pH 8.0)). Add 10 mL heat inactivated bovine serum and 1 mL of 0.5 M EDTA to 500 mL of PBS. Filter sterilize through a 0.2 &#181;m filter and store at 4 &#176;C. 
	NOTE: Solutions can be stored at 4 &#176;C for up to 1 month.</li>
</ol>
2. Labelling of Circulating Leukocytes
<ol>
	<li>Aseptically prepare anti-mouse CD45 injection with 1 &#181;g of antibody diluted in sterile phosphate buffered saline (PBS) for a final injection volume of 200 &#181;L/mouse. Load volume into a 28.5G insulin syringe and keep away from light. 
	NOTE: Use two different fluorochrome-labelled anti-CD45 antibodies in order to differentiate the intravascular (stained through i.v. administration) from the intracardiac staining (stained after digestion; see step 4).</li>
	<li>Pre-warm mice (8&#8211;12 weeks old) by exposing the tail to a red heat lamp for 5 min. 
	CAUTION: Do not overheat the animal. Keep the lamp at a safe distance (&#62;30 cm).
	<ol>
		<li>Restrain the animal in the sternal position using an appropriate device. Expose and stabilize the tail with the non-dominant hand by immobilizing the base of the tail with the index and middle finger and the mid-distal region of the tail with the thumb and ring finger. 
		CAUTION: Do not apply too much force at the tip of the tail as the skin can be pulled off. Insert the needle with the bevel facing up into the vein. Keep the needle parallel to the vein at all times as it is easy to puncture the vein. 
		NOTE: Blood entering the syringe may occur and is a sign of a good injection. Bending the needle to a 90&#176; angle can facilitate i.v. administration. Confirm with your safety office as bending needles may represent a hazard.</li>
		<li>Inject 200 &#181;L intravenously into the tail vein. The fluid should flow easily and temporarily clear the vein. 
		CAUTION: Do not force fluid in if resistance is met. This is sign of misplacement of the needle.</li>
		<li>Allow antibody to circulate for 5 min before humanely euthanizing mouse.</li>
	</ol>
	</li>
</ol>
3. Heart Isolating and Digestion
<ol>
	<li>Euthanize mice using CO2 or by other institutionally accepted euthanasia procedure.
	<ol>
		<li>For CO2 euthanasia, place the mouse in the CO2 chamber and set the fill rate to 20% of the chamber volume per minute (i.e., if the chamber is 10 L, fill at 2 L/min). Run for 2 min and monitor mouse until breathing has ceased.</li>
		<li>Shut off the CO2 and leave the mouse for an additional 1-2 min. Ensure breathing has ceased before opening the chamber and perform a toe pinch to confirm the mouse is dead before proceeding to process the mouse.</li>
	</ol>
	</li>
	<li>Spray the mouse with 70% ethanol. Lift the skin at the abdomen and dissect the mouse by cutting along the ventral midline beginning at the level of the hips up to the sternum. Open the abdomen and move the liver to expose the diaphragm.</li>
	<li>Cut the diaphragm, then the ribs on both sides of the midline, being careful to avoid puncturing the lungs and heart.
	<ol>
		<li>Expose the heart by peeling back the ribs, then cut first the left atria followed by the right atria. 
		&#8203;CAUTION: Use caution when cutting the atria, ensuring no arteries or veins are injured, as this can decrease the efficiency of flushing the blood contained in the organs.</li>
		<li>Perfuse the heart with 15&#8211;20 mL of cold PBS using a 60 mL syringe fitted with a 21 G needle and cold PBS. Gently grasp the base of the heart with forceps and insert the needle into the left ventricle near the apex. Repeat the procedure in the right ventricle. A proper injection results in white lungs and pale liver. 
		&#8203;NOTE: Perfusion should be performed within 5 minutes after euthanasia in order to avoid blood coagulation, which can contaminate the organs of interest with circulating cells.</li>
	</ol>
	</li>
	<li>Hold the base of the heart with forceps and gently pull the heart up, then detach the heart by cutting the major pulmonary arteries and veins, aorta and the pericardial sack.
	<ol>
		<li>Clean the heart by gently holding the heart between the thumb and index fingers in a clean lint-free paper towel and pull off the atria and remnants of major arteries and veins and any other contaminants with forceps. 
		&#8203;NOTE: Ensure the entirety of the atria is removed and no other tissues, such as the lung or blood vessels, are present as these tissues have their own resident immune cells. The lung in particular has a large immune cell population that can mask the smaller populations of those within the myocardium.</li>
	</ol>
	</li>
	<li>Place the heart in a 5 cm plastic dish with 50 &#181;L of cold PBS.
	<ol>
		<li>Mince the heart using curved dissecting scissors until there are no visible pieces and it has a smooth consistency. 
		&#8203;NOTE: Try to keep the heart in a contained area of the dish during chopping to minimize tissue loss.</li>
		<li>Transfer thetrituratedheart tissue using curved forceps into 2 mL microcentrifuge tube with 800 &#181;L cold Dulbecco&#39;s Modified Eagle Medium (DMEM).</li>
	</ol>
	</li>
	<li>Add 450 U/mL ofcollagenase I, 60 U/mL of hyaluronidase type I-S, and 60 U/mL of DNase-I to each sample.
	<ol>
		<li>Incubate samples at 37 &#176;C on a rocking shaker at 50 rpm for 45&#8211;60 min.</li>
	</ol>
	</li>
	<li>After digestion, briefly vortex samples (20 s) and immediately place on ice to keep samples at 4 &#176;C.
	<ol>
		<li>Using a 1 mL pipettor, pipette samples up and down until tissue samples are homogeneous and do not clog the pipette tip. 
		&#8203;NOTE: Defining a specific number of times to pipette may increase reproducibility. If the hearts are not optimally digested, pieces of myocardium may obstruct the pipette. Gently grinding the pipette tip against the microcentrifuge tube will help homogenize bigger pieces of tissue.</li>
	</ol>
	</li>
	<li>Pre-wet a 40 &#181;m cell strainer placed on a 50 mL conical tube with 1 mL of cold HBB.
	<ol>
		<li>Add homogenized sample to the filter and wash through by slowly pouring 12&#8211;14 mL of cold HBB.</li>
		<li>Transfer filtered samples to a labelled 15 mL conical tube and keep at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Centrifuge samples at 400 x g for 5 min at 4 &#176;C. Aspirate off supernatant.
	<ol>
		<li>Lyse red blood cells by adding 1 mL of Ammonium-Chloride-Potassium (ACK) lysis buffer to each sample. Resuspend samples by vortexing and incubate at room temperature for 5 min.</li>
		<li>Once lysis has completed, add 5&#8211;8 mL of cold Hank&#39;s Balanced Salt Solution (HBSS) to stop hemolysis.</li>
	</ol>
	</li>
	<li>Centrifuge samples at 400 x gfor 5 min at 4 &#176;C. Aspirate off supernatant.</li>
	<li>Add 1 mL of FACS buffer to each sample and transfer to a labelled 1.5 mL microcentrifuge tube. 
	NOTE: The protocol can be paused here for up to 2 h with samples stored at 4 &#176;C.</li>
</ol>
4. Flow Cytometry Staining and Analysis
<ol>
	<li>Centrifuge samples at 400 x g for 5 min at 4 &#176;C.
	<ol>
		<li>To reduce non-specific antibody binding, dilute 1:100 anti-CD16/CD32 and Monocyte Blocker (1:20) (see Table of Materials) accounting for 50 &#181;L FACS per sample. Incubate at room temperature for 15 min.</li>
		<li>Prepare the antibody dilution (1:250 for each antibody) accounting for 50 &#181;L of FACS per sample. 
		NOTE: A viability dye can be included in the antibody cocktail in order to exclude dead cells. Alternatively, small size exclusion by flow analysis can be used (Figure 1C and D).</li>
		<li>Without washing, add 50 &#181;L of antibody master mix to each sample and mix.</li>
		<li>Incubate samples at 4 &#176;C for 30 min in the dark.</li>
	</ol>
	</li>
	<li>Add 600 &#181;L FACS to each sample to wash and centrifuge samples at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate off supernatant and resuspend pellet in 300 &#181;L FACS. Keep samples at 4 &#176;C in the dark. 
	NOTE: Some viability dyes, such as DAPI, require staining within 10 min prior to running flow cytometry.</li>
	<li>Analyze by flow cytometry (see Figures 1 and 2) within the next 3 h. If a longer time is required, short term fixation using a low concentration paraformaldehyde (0.5&#8211;1% in PBS) is recommended to preserve staining. 
	NOTE: It is highly recommended to filter samples through a 40 &#181;m cell strainer prior to initiate flow cytometry analysis in order to prevent obstruction of the fluidics of the flow cytometer.</li>
</ol>

<ol>
	<li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+natural+history+of+human+myocarditis.">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li><li>Epelman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Pinto, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814 (2012).</li><li>Lavine, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+function+of+cardiac+macrophages.">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li><li>Hulsmans, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Nahrendorf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li><li>Clemente-Casares, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD103(+)+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis.">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li><li>Van der Borght, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+Infarction+Primes+Autoreactive+T+Cells+through+Activation+of+Dendritic+Cells.">Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.</a> Cell Reports. 18 (12), 3005-3017 (2017).</li><li>Galkina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+migration+of+effector+CD8++T+cells+into+the+interstitium+of+the+normal+lung.">Preferential migration of effector CD8+ T cells into the interstitium of the normal lung.</a> Journal of Clinical Investigation. 115 (12), 3473-3483 (2005).</li><li>Edelson, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+CD103++dendritic+cells+form+a+unified+subset+developmentally+related+to+CD8alpha++conventional+dendritic+cells.">Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells.</a> Journal of Experimental Medicine. 207 (4), 823-836 (2010).</li></ol>

The authors have nothing to disclose.

Myocardial inflammation, or myocarditis, is a feature of most cardiovascular diseases. However, the myocardium is not devoid of its own immune components in non-disease states. During steady state, many immune cells reside in the myocardium and play the essential roles of maintenance and protection. The characterization of these diverse populations of cells would not have been possible without methods such as the one presented in this protocol.
A combination of mechanical and enzymatic digesti...

Different forms of myocardial stress or injury, including ischemic (ischemia reperfusion or myocardial infarction) and non-ischemic (hypertension or myocarditis), promote the recruitment of inflammatory cells with reparative and protective, but also pathogenic properties. As early as 1891, Romberg first described the presence of cellular infiltrates in the myocardium of patients infected with typhus and scarlet fever1. However, the detailed study of cardiac immune cells required the development of more advanced immunophenotyping techniques. As a consequence, only recently have we started to understand that a diverse population of immune cells with essential maintenance roles during steady state resides within the myocardium.
Histology has been, and still is, the most common method to characterize cardiac immune cells during inflammation. However, while histology represents a valuable diagnostic tool, its use in the study of cardiac immune cells has important limitations. The immune cells residing in the myocardium represent a very small proportion of the total cells and are more or less evenly distributed in a proportionally immense space. Similarly, several forms of cardiac inflammation, such as viral myocarditis, show focal patterns of inflammation. This means that accurate analysis of the immune system of the heart often require higher degrees of histological sampling, increasing the cost to reduce bias. Moreover, histology provides a very limited amount of usable information in cell identification (e.g. number of parameters). With an ever increasing number of cell subsets that require the use of multiple expression markers (including surface markers, transcription factors or secreted molecules), flow cytometry has established itself as the most powerful tool for immunophenotyping, due to low-cost and high-throughput.
The use of immunophenotyping techniques is closely dependent on the development of efficient digestion protocols that allowed for single-cells analysis of large numbers of cells. The development of exhaustive protocols of cell extraction opened a new window of opportunities in the study of cardiac immunology. Through the combination of digestion, flow cytometry and transcriptomics, the major populations of macrophages and dendritic cells residing in the heart have been characterized2,3. The most abundant population of cardiac immune cells during steady state are CD64+MerTK+ macrophages, which can be further divided based on their expression of CCR2 or CD11c2. CCR2+ macrophages originate from adult bone marrow hematopoiesis, whereas the majority of CCR2- macrophages, which can express high or low levels of MHC-II, are mainly of prenatal origin. Macrophages perform key functions such as repair4 and removal of debris5 during injury or supporting electrical connectivity6 in steady state. During different forms of inflammation an important influx of Ly6Chi MHC-IIlo CD64int monocytes occur, which later differentiate into Ly6Chi CCR2+ macrophages2,7. A significantly smaller, but important, population of cells residing in the myocardium of healthy individuals is composed of dendritic cells8,9. The two major subsets of cardiac conventional DCs (cDCs) have been recently characterized: cDC1 (CD103+ DCs) and cDC2 (CD11b+ DCs). DCs play important roles in defense against infection8 but can also promote self-injury during inflammation, particularly during myocardial infarction9.
Here, we describe a simple method for isolation of viable cardiac immune cells from mouse myocardium. The method combines enzymatic and mechanical digestion with a cell-strainer filtration in order to obtain a single cell suspension that can be analyzed, or further purified, by flow cytometry sorting or magnetic bead enrichment. Accurate measurements of extravascular myocardial cells require cardiac perfusion in order to remove possible contaminants from the bloodstream of the cardiac microvasculature. In addition, we present an optional step of intravascular labeling of immune cells that can be used to further differentiate myocardial cells from intravascular contaminants, based on the Galkina et al. protocol10. Finally, we present a basic flow cytometry analysis to identify the major macrophage and cDC subpopulations.

<table><tbody><tr><td>Phosphate buffered saline</td><td>Wisent</td><td>311-010-CL</td><td>1x PBS</td></tr><tr><td>21 G x 1 1/2 (0.8 mm x 40 mm) PrecisionGlide Needle</td><td>BD</td><td>305167</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Wisent</td><td>311-511-CL</td><td>1x HBSS</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td></td></tr><tr><td>Bovine serum</td><td>Sigma</td><td>B9433</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetic acid</td><td>BioShop</td><td>EDT111</td><td></td></tr><tr><td>Vacuum filter , 0.2 &amp;micro;m Filtropur V50</td><td>Sarstedt</td><td>83.1823.001</td><td></td></tr><tr><td>28 G 1/2 1 cc insulin syringe</td><td>BD</td><td>329424</td><td></td></tr><tr><td>60 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Wisent</td><td>319-005-CL</td><td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td></tr><tr><td>Collagenase I</td><td>Sigma</td><td>C0130</td><td>from Clostridium histolyticum</td></tr><tr><td>Hyaluronidase type I-S</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>DNase-I</td><td>Sigma</td><td>D4513</td><td>from bovine pancreas</td></tr><tr><td>Cell strainer, 40 &amp;micro;m Nylon</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Ammonium-Chloride- Potassium (ACK) lysis buffer</td><td>Lonza</td><td>10-546E</td><td></td></tr><tr><td>Alexa Fluor 700 anti-mouse/human CD11b</td><td>Biolegend</td><td>101222</td><td>1:250 dilution</td></tr><tr><td>APC/Cy7 anti-mouse Ly-6c</td><td>Biolegend</td><td>128025</td><td>1:250 dilution</td></tr><tr><td>APC anti-mouse CD103</td><td>Biolegend</td><td>121414</td><td>1:250 dilution</td></tr><tr><td>Brilliant Violet 605 anti-mouse CD11c</td><td>Biolegend</td><td>117334</td><td>1:250 dilution</td></tr><tr><td>PE anti-mouse Ly-6G</td><td>Biolegend</td><td>127607</td><td>1:250 dilution</td></tr><tr><td>Pacific Blue anti-mouse I-Ab</td><td>Biolegend</td><td>116422</td><td>1:250 dilution</td></tr><tr><td>FITC anti-mouse CD64 (FcgRI)</td><td>Biolegend</td><td>139315</td><td>1:250 dilution</td></tr><tr><td>PE/Cy7 anti-mouse CD45</td><td>Biolegend</td><td>103113</td><td>1:250 dilution</td></tr><tr><td>PerCP/Cy5.5 anti-mouse CD45</td><td>Biolegend</td><td>103132</td><td>1:40 dilution</td></tr><tr><td>TruStain fcX (anti-mouse CD16/32)</td><td>Biolegend</td><td>101320</td><td>1:100 dilution</td></tr><tr><td>True-Stain Monocyte Blocker</td><td>Biolegend</td><td>426101</td><td>1:20 dilution</td></tr><tr><td>FlowJo V10</td><td>TreeStar Inc</td><td></td><td>https://www.flowjo.com/solutions/flowjo</td></tr><tr><td>Mouse: Batf3-/-</td><td>The Jackson Laboratory</td><td>JAX: 013755</td><td></td></tr></tbody></table>

isolation and identification of extravascular immune cells of the heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

To date, no good method has been designed to isolate immune cells from other cardiac cell components, such as cardiomyocytes. Hence, analysis of the cardiac single cell suspension by flow cytometry requires a pre-gating with CD45 to identify the immune cell populations, followed by single cell and small size exclusion gating (Figure 1A). Alternatively, a viability staining can be performed to exclude dead cells. Small size exclusion and viabi...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.5K Views.  University Health Network (UHN). This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Video: Isolation and Identification of Extravascular Immune Cells of the Heart

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Developmental Biology

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Medicine

Revealing Murine Pulmonary Veins Myocardial Architecture

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

Author Spotlight: Developing a Translational Model for Atrial Fibrillation Research Across Species 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Isolation and Identification of Extravascular Immune Cells of the Heart

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Isolation of Functional Cardiac Immune Cells

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Immunomagnetic Isolation of the Vascular Wall-Resident CD34⁺ Stem Cells from Mice