Name: isolation and identification of extravascular immune cells of the heart
Uploaded: 2018-08-23T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

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 f...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;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 height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

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