Name: isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node
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Description: Watch this Scientific Journal Video about Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node at JoVE.com

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>

<ol>
	<li>Nikolaidou, T., Aslanidi, O. V., Zhang, H., Efimov, I. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+function+of+the+cardiac+conduction+system.">The formation and function of the cardiac conduction system.</a> Development. 143 (2), 197-210 (2016).</li><li>Ye Sheng, 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=Isolation+and+characterization+of+atrioventricular+nodal+cells+from+neonate+rabbit+heart.">Isolation and characterization of atrioventricular nodal cells from neonate rabbit heart.</a> Circulation: Arrhythmia and Electrophysiology. 4 (6), 936-946 (2011).</li><li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723-737 (2011).</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=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><tbody><tr><td>&lt;strong&gt;Anesthesia&lt;/strong&gt;</td><td></td><td></td><td></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>&lt;strong&gt;&lt;em&gt;In vivo&lt;/em&gt; instrumentation&lt;/strong&gt;</td><td></td><td></td><td></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>&lt;strong&gt;General lab instruments&lt;/strong&gt;</td><td></td><td></td><td></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>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></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>&lt;strong&gt;Flow cytometry machine&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beckman Coulter</td><td>Beckman coulter</td><td>MoFlo Astrios</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FlowJo v10</td><td>FlowJo</td><td></td><td></td></tr><tr><td>&lt;strong&gt;General Lab Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;micro;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>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></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&#039;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 &amp;minus; 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>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fentanyl 0.5&amp;nbsp;mg/10&amp;nbsp;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>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></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>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></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

4.1K Views.  Ludwig-Maximilian-Unversity Munich (LMU). We beschrijven een protocol voor microdissectie en daaropvolgende isolatie van cardiale resident microfagen door fluorescentie-geactiveerde celsortering specifiek van sinus en AV-knoop in de muis. Met de precieze microdissectie van de sinus en de V-knoop, gevolgd door de combinatie van magnetische en fluorescentie-geactiveerde celsortering, zijn we in staat om residente macrofagen specifiek te isoleren van de sinus en de V-knoop met hoge zuiverheid. Identificatie en isolatie van cardiale macr...

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 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 ...

Immunology and Infection

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.

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 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 ...

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 ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Biology

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Sinoatrial Node Isolation from Mouse Model: A Procedure to Harvest SA Node from Murine Heart

Begin by holding the skin of the euthanized mouse with a hemostat, and use surgical scissors to make a transverse incision in the skin, just beneath the bottom of the rib cage from the left costal arch to the right costal arch. Use surgical scissors to cut open the peritoneum, and carefully separate the liver from the diaphragm, without nicking the liver, to prevent excessive bleeding.
Incise the diaphragm along the thorax to expose the thoracic cavity. Use the surgical scissors to cut the lateral walls of the rib cage, from the edges of the costal arches up to the clavicles, to expose the heart. Then, use a 23-gauge syringe needle to pin the rib cage over the shoulder.
Use a transfer pipette to drop warm heparinized Complete Tyrode&#39;s solution on the heart to keep it moist. Hold the lungs with extra fine Graefe forceps, and sever the trachea with surgical scissors to remove the lungs. For removing the heart, hold the apex of the heart with extra fine Graefe forceps, and cut the aorta and inferior vena cava with surgical scissors.
Transfer the heart to a Petri dish containing a cured silicone elastomer, and use a transfer pipette for bathing the heart with 2 to 3 milliliters of warm heparinized Complete Tyrode&#39;s solution. Attach the apex of the heart to the dish with a dissection pin.
Hold the inferior vena cava with Dumont #2 laminectomy forceps. Insert a 22-gauge syringe needle through the inferior and superior vena cava to locate their position in the right atrium, which also identifies the approximate position of the sinoatrial node.
Hold the right atrial appendage with Dumont #2 laminectomy forceps, and put a dissection pin through the right atrial appendage to hold it in place. Repeat the same procedure for the left atrial appendage. Then, remove the syringe needle that spans the vena cava.
Use Castroviejo scissors to remove the apex of the heart by making a transverse incision across the ventricles, or releasing the blood from the heart. Then, wash the heart by adding warm heparinized Complete Tyrode&#39;s solution. Use Castroviejo scissors to cut along the atrioventricular septum until the atria are separated from the ventricles, and keep the incision closer to the ventricle.
Cut along the interatrial septum to remove the left atrium. Place the dissection pins in the periphery of the right atrium to make it lay flat. Remove any remaining fat, vessels, or tissue from the atrium using the Castroviejo scissors, and locate the sinoatrial node in the right atrium.

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

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