This work was supported by the China Scholarship Council (CSC, to R. Xia), the German Centre for Cardiovascular Research (DZHK; 81X2600255 to S. Clauss, 81Z0600206 to S. K&#228;&#228;b, 81Z0600204 to C.S.), the Corona Foundation (S199/10079/2019 to S. Clauss), the SFB 914 (project Z01 to S. Massberg), the ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 to S. Clauss) and the Heinrich-and-Lotte-M&#252;hlfenzl Stiftung (to S. Clauss). The funders had no role in manuscript preparation.

Animal care and all experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics committee of the University of Munich and all the procedures undertaken on mice were approved by the Government of Bavaria, Munich, Germany. C57BL6/J mice were commercially obtained.
1. Preparations
<ol>
	<li>Prepare Cell sorting buffer (Table 1) and store at 4 &#176;C. 
	NOTE: During the whole experimental procedure, the cell sorting buffer should always be on ice.</li>
	<li>Prepare Digestion buffer (Table 1) shortly before the digestion as the activity of collagenase could only be detected for few hours at room temperature.</li>
	<li>Refer to the previously published protocol for the preparation of dissection dish10. In brief, add 30 mL of agarose gel (3%-4%) into a 100 mm diameter Petri dish and cool down at room temperature.</li>
</ol>
2. Animal sacrifice and heart excision
<ol>
	<li>Anesthetize the mouse with isoflurane by placing it into an incubation chamber connected with an isoflurane vaporizer and flushed with 5% isoflurane/95% oxygen.</li>
	<li>After the injection of fentanyl for analgesia, open the rib cage and perfuse the heart by injecting 5-10 mL of ice-cold 1x PBS directly into the left ventricle (LV). Extract the mice heart and put it on the dissection dish. Experimental details have been described in detail previously10.</li>
</ol>
3. Microdissection of SAN and AVN
<ol>
	<li>After isolating the heart, perform the following microdissection procedures in the dissection dish with ice-cold 1x PBS under the dissecting microscope.</li>
	<li>Use the cardiac anatomical landmarks, i.e., aorta, pulmonary artery, coronary sinus, left/right ventricle, etc. to determine the left/right (left: LV; right: RV) and anterior/posterior (anterior: aorta; posterior: coronary sinus) of the heart. After the orientation is determined, turn around the heart with the front of it at the bottom of the dish (to expose the large veins that are located posterior).</li>
	<li>Microdissection of SAN 
	NOTE: Microdissection of the SAN have been previously described10. The process is described in brief below.
	<ol>
		<li>Expose the inter-caval region by pining the right atrial appendages (RAA) and the tissue adjacent to superior vena cava (SVC) and inferior vena cava (IVC) on the microdissection dish using insect pins.</li>
		<li>Cut the heart along the interatrial septum parallel to the crista terminalis (CT) to separate the inter-caval region and to obtain the SAN sample (Figure 1A, Figure 2A). Put the sample in an empty 1.5 mL microcentrifuge tube on ice.</li>
	</ol>
	</li>
	<li>Microdissection of AVN
	<ol>
		<li>After collection of the SAN sample ensure that the RAA and parts of the right atrium (RA) have already been cut away leaving only the interatrial septum (IAS) and, interventricular septum (IVS).</li>
		<li>Pin the remaining parts of the heart through the tissue adjacent to the IAS and IVS using insect pins to make the right atrial side of IAS facing up.</li>
		<li>Look at the right atrium on the endocardial surface for the triangle of Koch. It will be bordered anteriorly by the hinge-line of the septal leaflet of the tricuspid valve (TV), and posteriorly by the tendon of Todaro. The orifice of the coronary sinus is observed at the base. (Figure 1B, Figure 2B).</li>
		<li>Cut the triangle of Koch, which contains the AVN, and directly put it in an empty 1.5 mL microcentrifuge tube on ice.</li>
	</ol>
	</li>
</ol>
4. Digestion
<ol>
	<li>Prepare the Digestion buffer (Table 1) shortly before use.</li>
	<li>Mince the SAN and AVN tissue well with scalpels. 
	NOTE: Mincing the tissue well will increase the digestion efficiency and help to get good cell suspension for sorting. As the SAN and AVN samples are quite small, mincing the tissue directly inside the 1.5 mL microcentrifuge tube is recommended to reduce the loss of sample.</li>
	<li>Add 500 &#181;L of digestion buffer per sample and wash down all minced tissue from the wall of the 1.5 mL microcentrifuge tube. Gentle pipetting helps digesting the sample.</li>
	<li>Homogenize the tube on a vortex machine (settings: 37 &#176;C, 750 rpm for 1 h).</li>
	<li>After digestion, transfer the tissue suspension to a fresh 15 mL centrifuge tube by passing through a 40 &#181;m cell strainer. Rinse the cell strainer with an additional 5 mL of cell sorting buffer to stop the digestion.</li>
	<li>Centrifuge the 15 mL tube at 350 x g for 7 min at 4 &#176;C. Then remove the supernatant completely using the pipette. Resuspend the cell pellet with 90 &#181;L cell sorting buffer. 
	&#8203;NOTE: Before magnetic separation, pipette the cell suspension gently a few times or pass through a 30 &#181;m cell strainer to remove cell clumps if necessary, to obtain a single cell suspension for optimal performance of magnetic enrichment of interesting cell populations.</li>
</ol>
5. Magnetic enrichment of CD45 and sample staining
NOTE: To isolate the cardiac macrophages with high sorting efficiency, exclusion of undesired cells including lymphocytes was performed with CD45 microbeads according to the manufacturer&#39;s protocol. Based on the sorting panel, cardiac resident macrophages were identified as CD45highCD11bhighCD64high Ly6Clow/int F4/80high.
<ol>
	<li>Add 10 &#181;L of CD45 microbeads per 107 total cells to the cell suspension in the 15 mL centrifuge tube. Mix the samples well and incubate them for 15 min at 4 &#176;C. 
	NOTE: The cell counting using a hemocytometer should be briefly done to make sure that each tube contains no more than 107 total cells. When working with higher cell numbers, the volume of magnetic beads needs to be scaled up.</li>
	<li>Prepare the antibody mixture by diluting the following antibodies in cell sorting buffer (1:100 dilution for each antibody): CD45-PE, CD11b-APC-Cy7, CD64-APC, F4/80-PE-Cy7, Ly6C-FITC. DAPI will be added later to the staining for live/dead discrimination.</li>
	<li>After 15 min of magnetic bead incubation, add 100 &#181;L of antibody mixture directly into the cell suspension in the 15 mL tube (then all the antibodies&#39; final concentration is 1:200) and incubate for 20 min at 4 &#176;C.</li>
	<li>After 20 min of antibody incubation, wash the cell suspension by adding 1-2 mL of cell sorting buffer per 107 cells and centrifuge at 350 x g for 10 min. Completely remove the supernatant by pipetting.</li>
	<li>Resuspend up to 108 cells in 500 &#181;L of cell sorting buffer. 
	NOTE: The maximum cell number for magnetic separation should be determined according to the manufacturer&#39;s protocol.</li>
	<li>Prepare the magnetic separation set.
	<ol>
		<li>Attach the magnetic column to a suitable magnetic separator and place a collection tube under the magnetic column.</li>
		<li>Prepare the magnetic columns by rinsing with cell sorting buffer: add 500 &#181;L of cell sorting buffer at the top of the column and let the buffer pass through.</li>
	</ol>
	</li>
	<li>Apply the cell suspension immediately onto the column while the cell sorting buffer is passing through. 
	NOTE: Avoid the formation of air bubbles in the column. As per the manufacture&#39;s protocol, although the column filling time might change from storage conditions, it has no influence on the quality of the separation.</li>
	<li>Wash the column with 3 x 500 &#181;L cell sorting buffer. The flow-through in step 5.7 and step 5.8 contain unlabeled cells, which can be discarded if no further experiment is needed. 
	&#8203;NOTE: Add the cell sorting buffer immediately when the column reservoir is nearly empty.</li>
	<li>Remove the column from the magnetic separator and place it on a new collection tube.</li>
	<li>Add 1 mL of cell sorting buffer onto the column. Immediately flush the column by firmly applying the plunger supplied with the column. The flow-through contains the magnetically labeled cells.</li>
	<li>Add DAPI solution into all the collected magnetically labeled cell suspensions shortly before running them on the cell sorter. Adjust the final concentration of DAPI to 0.3-0.5 &#181;g/mL.</li>
	<li>Perform FACS analysis.</li>
</ol>
6. Samples for compensation
<ol>
	<li>Prepare 6 brown 1.5 mL microcentrifuge tubes labeled as &#34;PE&#34;, &#34;APC-Cy7&#34;, &#34;APC&#34;, &#34;PE-Cy7&#34;, &#34;FITC&#34;, and &#34;DAPI&#34; respectively to protect antibodies from light. Prepare one more 1.5 mL microcentrifuge tube labeled as &#34;unstained&#34;. 
	NOTE: This could be done at the same time as incubating the cell suspension with the microbeads and antibodies. The unstained sample could be the cardiomyocyte tissue collected randomly from the spared heart tissue and also treated according to step 4.</li>
	<li>Dilute each single fluorescence-conjugated antibody with cell sorting buffer into 1:50 in the 1.5 mL brown microcentrifuge tubes that are marked accordingly.</li>
	<li>Add one drop of compensation beads solution and incubate for 20 min at 4 &#176;C.</li>
	<li>Add 2 mL of cell sorting buffer into each 1.5 mL brown microcentrifuge tube and centrifuge at 450 x g for 5 min. Completely discard the supernatant and resuspend the bead containing pellet with 300 &#181;L cell sorting buffer and transfer them into cell sorter tubes that are also marked accordingly.</li>
</ol>
7. Running on the cell sorter and gating strategy
<ol>
	<li>Apply the unstained sample and compensation tubes first and adjust the voltages of each channel to align both the positive and negative peak to the proper position of the axis. Save the compensation settings and apply it to the following samples.</li>
	<li>Apply the samples on the cell sorter. Set the gating strategy as described in Figure 4. Cardiac resident macrophages are identified as CD45highCD11bhighCD64highLy6Clow/int F4/80high. DAPI is used as a cell viability marker.</li>
	<li>Check the flow cytometry charts to confirm that the cell population of interest is properly shown on the charts. If not, adjust the voltage of each channel to the center view of each chart.</li>
	<li>If the voltage settings are satisfactory, start the sorting procedure. Collect the sorted cell population into culture medium composed of DMEM containing 10% fetal bovine serum, supplemented with 100 &#181;g/mL streptomycin and 100 U/mL penicillin.</li>
</ol>
8. Resident macrophages culture
<ol>
	<li>After gathering the sorted macrophages, transfer the cells immediately either to 35 mm tissue culture dishes or 24 well plate, or directly use them for subsequent experiments.</li>
	<li>To culture sorted macrophages, incubate the cells at 37 &#176;C, 5% CO2 incubator.</li>
	<li>Change the culture medium every 48-72 h. Floating dead cells can be easily removed by medium change. Use live macrophages attaching to the bottom of culture dish for subsequent experiment.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationship+in+the+sinus+and+atrioventricular+nodes.">Structure-function relationship in the sinus and atrioventricular nodes.</a> Pediatric Cardiology. 33 (6), 890-899 (2012).</li><li>Clauss, 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=Animal+models+of+arrhythmia:+classic+electrophysiology+to+genetically+modified+large+animals.">Animal models of arrhythmia: classic electrophysiology to genetically modified large animals.</a> Nature Review Cardiology. 16 (8), 457-475 (2019).</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>Rosas, 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+transcription+factor+Gata6+links+tissue+macrophage+phenotype+and+proliferative+renewal.">The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal.</a> Science. 344 (6184), 645-648 (2014).</li><li>Schulz, C., 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+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</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>Gordon, S., Pluddemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+macrophages:+heterogeneity+and+functions.">Tissue macrophages: heterogeneity and functions.</a> BMC Biology. 15 (1), 53 (2017).</li><li>Bajpai, G., 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+human+heart+contains+distinct+macrophage+subsets+with+divergent+origins+and+functions.">The human heart contains distinct macrophage subsets with divergent origins and functions.</a> Nature Medicine. 24 (8), 1234-1245 (2018).</li><li>Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages.">Tissue-resident macrophages.</a> Nature Immunology. 14 (10), 986-995 (2013).</li><li>Xia, 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=Whole-mount+immunofluorescence+staining,+confocal+imaging+and+3D+reconstruction+of+the+sinoatrial+and+atrioventricular+node+in+the+mouse.">Whole-mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse.</a> Journal of Visualized Experiments. (166), e62058 (2020).</li><li>van Weerd, J. H., Christoffels, V. 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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=Revisiting+cardiac+cellular+composition.">Revisiting cardiac cellular composition.</a> Circulation Research. 118 (3), 400-409 (2016).</li><li>Bajpai, G., 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=Tissue+resident+CCR2-+and+CCR2++cardiac+macrophages+differentially+orchestrate+monocyte+recruitment+and+fate+specification+following+myocardial+injury.">Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.</a> Circulation Research. 124 (2), 263-278 (2019).</li><li>Honold, L., Nahrendorf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+monocyte-derived+macrophages+in+cardiovascular+disease.">Resident and monocyte-derived macrophages in cardiovascular disease.</a> Circulation Research. 122 (1), 113-127 (2018).</li><li>Leid, 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=Primitive+embryonic+macrophages+are+required+for+coronary+development+and+maturation.">Primitive embryonic macrophages are required for coronary development and maturation.</a> Circulation Research. 118 (10), 1498-1511 (2016).</li></ol>

No potential conflict of interest relevant to this article was reported.

In this manuscript, we describe a protocol for the enrichment of cardiac resident macrophages specifically from the SAN and AVN regions at high purity.
Macrophages are divided into subpopulations based on their anatomical location and functional phenotype. They can also switch from one functional phenotype to another in response to variable microenvironmental signals13. Compared to other organs such as bone marrow and liver, cardiac tissue contains a lower percentage of immune cells and lower absolute numbers of each cellular subpopulation14. Therefore, cell sorting, enrichment and purification methods are necessary tools to obtain sufficient amounts of the cell population of interest. Fluorescence-activated cell sorting, and MACS allow to obtain pure, sorted cell populations as it permits simultaneous measurement of various properties of the cells.
Different flow cytometry panels have been described for the identification of subpopulations of cardiac macrophages3,6,15. The function of macrophages in steady state and disease not only depend on their developmental origin but also on the tissue environment. In general, the adult heart contains two major subsets of Ly6Clow/CCR2- resident macrophages that express different levels of MHC-II and which can maintain themselves via local proliferation at steady state whereas during disease classical Ly6Chigh monocytes are recruited to sites of inflammation, where they differentiate into macrophages16. During development, different subpopulations of macrophages occupy different cardiac locations associated with distinct functions17. We aimed to study the resident macrophages specifically from the cardiac conduction system, especially the resident macrophages from the SAN and AVN regions. According to Hulsmans et al. cardiac resident macrophages are identified as CD45high CD11bhighCD64highF4/80highLy6Clow/int.
The harvest of cardiac resident macrophages from one adult mouse for cell sorting requires approximately 3 hours. It is important to arrange the experimental procedures logically and to allow incubation in parallel to save time and to minimize handling of the possibly fragile macrophages in the suspension. As the sorting procedure could exert pressure on the sorted cells, we recommended to reduce the sorting time by using magnetic beads which could increase the sorting efficiency tremendously and also allows to obtain higher purity of resident macrophages.
The application of this protocol includes but is not limited to purification of macrophages and/or any other non-cardiomyocyte cell type from the cardiac tissue and any other mice strains. The sorted macrophages could be used for subsequent experiments, for example cell motility assays, gene or protein expression studies, etc. Single-cell RNA sequencing is also possible by collecting cells one-by-one directly from the collecting tube of the cell sorter.
However, flow cytometry-based cell sorting has its limitations. A precisely designed antibody panel is important and must consider the expression of antigens on the cell population of interest and the fluorophore conjugated to the antibodies. Cells function and viability might be altered by the binding antibodies, which might affect the outcomes of subsequent experiments. In addition, the complex cell sorting instruments are expensive, sophisticated, and also prone to problems with fluidics system blockages and laser calibration. Hence maintenance by highly trained specialist and properly operation by an experienced professional technician are required. Even though cell sorting could provide a pure cell population of interest, the overall efficiency is still relatively low.

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62236">Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node</a>. The Authors section was updated from:
Ruibing Xia123 
Simone Loy1 
Stefan K&#228;&#228;b13 
Anna Titova1 
Christian Schulz123 
Steffen Massberg123 
Sebastian Clauss123 
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-Unversity Munich (LMU) 
2Walter Brendel Center of Experimental Medicine (WBex) LMU Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)
to:
Ruibing Xia123 
Simone Loy1 
Stefan K&#228;&#228;b13 
Anna Titova1 
Christian Schulz123 
Steffen Massberg123 
Sebastian Clauss123 
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-University Munich (LMU) 
2Insitute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig-Maximilians-University (LMU) 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62236">Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node</a>. The Authors section was updated.

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

The sinoatrial node (SAN) physiologically initiates the electrical impulse and is, therefore, the primary pacemaker of the heart. The atrioventricular node (AVN) conducts the electrical impulse from the atria to the ventricles and also acts as a subsidiary pacemaker1. In general, generation and conduction of electrical impulses is a complex process that can be modulated by various factors2, including resident macrophage in SAN/AVN regions. A recent study by Hulsmans et al. demonstrates a specific population of cardiac resident macrophages which are enriched in the AVN and function as key players in keeping a steady heartbeat3. They found that macrophages are electrically coupled to the cardiomyocytes and could change the electrical properties of coupled cardiomyocytes. The authors also note that such conducting cells interleaving with macrophages are also present in other components of the cardiac conduction system, such as the SAN.
Currently, it is not fully known if the phenotype of resident cardiac macrophages differs between the cardiac regions. However, it has been shown that the tissue microenvironment can affect transcription and proliferative renewal of tissue macrophages4. Furthermore, since the cardiomyocyte phenotype has been demonstrated to be different between regions, the functional effects of macrophages on cardiomyocytes may also be region-specific, even if the macrophage phenotype itself may be the same. Therefore, further studies on specific cardiac regions are needed.
Recent studies have demonstrated that, at steady state, the tissue resident macrophages are established prenatally, arising independently of definitive hematopoiesis, and persist into adulthood5. However, after macrophage depletion or during cardiac inflammation, Ly6chi monocytes contribute to replenish cardiac macrophage population6. Studies involving genetic lineage tracing, parabiosis, fate mapping, and cell tracking showed the coexistence of a variety of tissue resident macrophages populations in organs and tissues, and, also, different cellular behavior of macrophage subsets that are potentially associated with their ontogeny7,8,9.
Characterization of resident cardiac macrophages has benefited from the use of magnetic activated cell sorting (MACS) and fluorescent activated cell sorting. These methods are particularly useful for isolating specific cell populations from multiple tissue fractions by labeling them with their cell surface markers. This not only leads to a higher purity of the isolated immune cell type, but also allows for phenotypic analysis. Here, we present a protocol including magnetic beads-coated cells followed with fluorescent activated cell sorting for the enrichment of cardiac resident macrophages specifically isolated from the SAN and AVN region.
To explore the characteristics of cardiac resident macrophages in conduction system and their function for cardiac conduction and arrhythmogenesis, precise localization and dissection of SAN and AVN are critical. For microdissection of SAN and AVN, anatomical landmarks are used for the region identification10. In brief, SAN is located at the junction of the superior vena cava and right atrium. AVN is located within the triangle of Koch, which is anteriorly bordered by the septal leaflet of the tricuspid valve, and posteriorly by the tendon of Todaro11. We also provide an accurate microdissection procedure of SAN and AVN in mice which is confirmed by histology and immunofluorescence staining.
Isolated resident macrophages could be used for further experiments such as RNA sequencing or could be recovered and cultivated for more than two weeks allowing various in vitro experiments. Therefore, our protocol describes a highly valuable procedure for the immuno-rhythmologist. Table 1 shows the composition of all the solutions needed, Figure 1 shows the microdissection landmarks for SAN and AVN. Figure 2 is schematic illustration of SAN and AVN localization. Figure 3 shows the histological staining of SAN and AVN (Masson&#39;s trichrome and immunofluorescence staining). Figure 4 shows a step-by-step gating strategy to isolate cardiac resident macrophages by fluorescence-activated cell sorting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer system</td>
			<td>Hugo Sachs Elektronik</td>
			<td>34-0458, 34-1030, 73-4911, 34-0415, 73-4910</td>
			<td>Includes an induction chamber, a gas evacuation unit and charcoal filters</td>
		</tr>
		<tr>
			<td>Modified Bain circuit</td>
			<td>Hugo Sachs Elektronik</td>
			<td>73-4860</td>
			<td>Includes an anesthesia mask for mice</td>
		</tr>
		<tr>
			<td>Surgical Platform</td>
			<td>Kent scientific</td>
			<td>SURGI-M</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">In vivo instrumentation</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue pins</td>
			<td>Fine Science Tools</td>
			<td>26007-01</td>
			<td>Could use 27G needles as a substitute</td>
		</tr>
		<tr>
			<td colspan="4">General lab instruments</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Sunlab</td>
			<td>D-8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette,volume 10ul, 100ul, 1000ul</td>
			<td>Eppendorf</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RH basic</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Microscopes</td>
		</tr>
		<tr>
			<td>Dissection stereo- zoom microscope</td>
			<td>vwr</td>
			<td>10836-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica microsystems</td>
			<td>Leica DM6</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Flow cytometry machine</td>
		</tr>
		<tr>
			<td>Beckman Coulter</td>
			<td>Beckman coulter</td>
			<td>MoFlo Astrios</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>FlowJo v10</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">General Lab Material</td>
		</tr>
		<tr>
			<td>0.2 &#181;m syringe filter</td>
			<td>sartorius</td>
			<td>17597</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>BD Microlance 3</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Tube</td>
			<td>FALCON</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Thermo scientific</td>
			<td>7632160</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Eppendorf</td>
			<td>30121872</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Sigma</td>
			<td>20-158</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>100063</td>
			<td>Component of TEA</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>850070</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1X) Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>F2442-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin &#8722; Streptomycin</td>
			<td>Sigma</td>
			<td>P4083</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Drugs</td>
		</tr>
		<tr>
			<td>Fentanyl 0.5&#160;mg/10&#160;ml</td>
			<td>Braun Melsungen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 1 ml/ml</td>
			<td>Cp-pharma</td>
			<td>31303</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen 5L</td>
			<td>Linde</td>
			<td>2020175</td>
			<td>Includes a pressure regulator</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6C FITC (clone HK1.4)</td>
			<td>BioLegend</td>
			<td>Cat# 128006</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 PE/Cy7(clone BM8)</td>
			<td>BioLegend</td>
			<td>Cat# 123114</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD64 APC (clone X54-5/7.1)</td>
			<td>BioLegend</td>
			<td>Cat# 139306</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b APC/Cy7(clone M1/70)</td>
			<td>BioLegend</td>
			<td>Cat# 101226</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PE (clone 30-F11)</td>
			<td>BioLegend</td>
			<td>Cat# 103106</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>diluted to 1:1000</td>
		</tr>
		<tr>
			<td colspan="4">Animals</td>
		</tr>
		<tr>
			<td>Mouse, C57BL/6</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We describe a practical procedure for the isolation of cardiac resident macrophages specifically from the SAN and AVN region. To confirm a correct dissection, Masson&#39;s Trichrome staining and immunofluorescent HCN4-staining is performed (Figure 3)12. With this protocol, we could collect approximately 60,000 macrophages from one whole heart. Figure 4 shows the gating strategy for sorting cardiac macrophages. Live resident cardiac macrophages were identified as CD45+CD11b+F4/80+CD64+Ly6C-. Figure 5 shows freshly sorted cardiac macrophages which were identified by their surface antigens CD45, F4/80 and CD11b. Freshly sorted cells were observed under brightfield view of microscope (Figure 5A). The sorted cells were positive for CD45 (CD45+) when observed under the fluorophore-PE channel (Figure 5B).&#160;The same view of the sorted cells when observed under fluorophore-APC-Cy7 channel showed CD11b+ phenotype (Figure 5C). The same view of the sorted cells when observed under fluorophore-PE-Cy7 channel showed F4/80+ phenotype (Figure 5D). Figure 5E is&#160;the merged image obtained with the fluorescent microscope for the sorted cells. These triple positive cells were identified as cardiac resident macrophages. Figure 6 shows isolated cardiac macrophages cultured in medium up to 6 days. White arrows indicate macrophages, black arrows indicated floating round-shape dead cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62236/62236fig01.jpg" /> 
Figure 1: Anatomy of the SAN and AVN under the dissection microscope. (A)&#160;Anatomy of the SAN under the dissection microscope. The location of the SAN is indicated by red dashed line within the inter-caval region (black dashed lines). (B) Anatomy of the AVN under the dissection microscope. This figure has been modified from previously published article10. The AVN (red dashed circle) is located at the apex of the triangle of Koch (white dashed triangle) near the bottom of the membranous septum. The triangle of Koch is formed by the tendon of Todaro (TT, green dashed line), tricuspid valve (TV, blue dashed line) and the orifice of the coronary sinus (CS, yellow dashed line). SVC, superior vena cava; IVC, inferior vena cava; IAS, interatrial septum; RA, right atrium; RAA, right atrial appendage; RV, right ventricle; CT, Crista terminalis; IVS, interventricular septum; OF, oval fossa. PA, pulmonary artery; RV, right ventricle; LV, left ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62236/62236fig02.jpg" /> 
Figure 2: Schematic illustration of SAN and AVN localization. (A) schematic illustration of SAN localization. SAN is indicated by a red dashed circle within the inter-caval region besides the CT (black dashed line). (B)&#160;schematic illustration of AVN localization. AVN is indicated by a red dashed circle inside the Koch triangle (grey dashed triangle) composed of TV and the orifice of CS. SVC, superior vena cava; IVC, inferior vena cava; IAS, interatrial septum; RA, right atrium; RAA, right atrial appendage; CT, Crista terminalis; IVS, interventricular septum; OF, oval fossa; IVS, interventricular septum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62236/62236fig03.jpg" /> 
Figure 3: Identification of the SAN and AVN with histological stain. Identification of the SAN (A,B) and AVN (C,D). Immunofluorescent staining of HCN4 positive conduction system cells in SAN (A) and AVN (C) as well as Masson&#39;s trichrome staining of SAN (B) and AVN (D). Red arrows indicate sinus node artery (SNA, B), black arrow and black dashed line indicate compact AVN, blue arrow indicates the central fibrous body (CFB).&#160;CT, crista terminalis; CFB, central fibrous body; CN, compact AVN; RA, right atrium; RV, right ventricle; IAS, interatrial septum; IVS, interventricular septum; TV, tricuspid valve; MV, mitral valve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62236/62236fig04.jpg" /> 
Figure 4: Gating strategy for cell sorting of resident cardiac macrophages. Mononuclear cells are identified, doublets are excluded by FSC-W vs. FSC-A and dead cells are excluded by DAPI (A-D). Live cells are gated on CD45+ leukocytes (E), and then gated on CD11b+ myeloid cells (F). Cardiac macrophages were identified by the expression of both F4/80 and CD64 (G), and then finally stratified by Ly6C expression (H). Live resident cardiac macrophages are identified as CD45+CD11b+F4/80+CD64+Ly6C-.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62236/62236fig05.jpg" /> 
Figure 5: Freshly sorted cardiac macrophages and immunofluorescent staining. (A) Freshly sorted cardiac macrophages. Immunofluorescent staining of specific surface antigens such as CD45 (B), CD11b (C), or F4/80 (D). According to the gating strategy, cardiac macrophages are identified as triple positive cells (E). Scale bar represents 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62236/62236fig06.jpg" /> 
Figure 6: Culture of sorted macrophages. Culture of sorted macrophages in culture medium for 48h (A, B), 96 h (C,D) and 6 days (E,F) respectively. Two individual culture dishes per time point are shown (dish 1: A, C, E; dish 2: B, D, F). White arrows indicate live macrophages with spindle-like shape and typical protrusions3. Black arrows indicate floating round-shape dead cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62236/62236fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Final concentration</td>
			<td>g or ml required</td>
		</tr>
		<tr>
			<td colspan="3">FACS buffer</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.50%</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>2 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1X)</td>
			<td></td>
			<td>500ml</td>
		</tr>
		<tr>
			<td colspan="3">Digestion buffer</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>450 U/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>125 U/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>60 U/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>60 U/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>20 &#181;l per 1 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1X)</td>
			<td>Add up to 1 ml for 2 samples</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td>79 ml</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>1%</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>20%</td>
			<td>20 ml</td>
		</tr>
		<tr>
			<td colspan="3">TAE (50x)</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>24.20%</td>
			<td>24.2 g</td>
		</tr>
		<tr>
			<td>100 % acetic acid</td>
			<td>5.71%</td>
			<td>5.71 ml</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>0.05 M</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td></td>
			<td>Add up to 100ml</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of solutions needed.

Watch this Scientific Journal Video about Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node at JoVE.com

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

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 ...

isolation-culture-resident-cardiac-macrophages-from-murine-sinoatrial

Nanyang Technological University

Research

JoVE Journal

Medicine

4.0K Views.  Ludwig-Maximilian-Unversity Munich (LMU). 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.

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

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

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Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Location, Dissection, and Analysis of the Murine Stellate Ganglion

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice