This work was supported by the Natural Science Funds of Zhejiang Province (LQ22H020010).

All the experiments were conducted in accordance with the guidelines for the care and use of laboratory animals at Zhejiang University and were approved by the Animal Advisory Committee at Zhejiang University.
1. Left anterior descending coronary artery ligation (LAD ligation) surgery
NOTE: Eight week old male C57BL/6J mice were used as models. The hearts were harvested 2 weeks after MI. Left anterior descending coronary artery ligation surgery was carried out as previously described and demonstrated11.
<ol>
	<li>Disinfect the surgical instruments with 70% ethanol before surgery.</li>
	<li>Weigh the mouse, and then anesthetize the mouse with intraperitoneal injections of 1% pentobarbital sodium (50 mg/kg). Observe the toe-pinch reflex, and measure the respiratory rate to evaluate the depth of anesthesia during the procedure.</li>
	<li>Place the mouse on the operating table with a 37 &#176;C heating pad. Use ophthalmic ointment to prevent eye dehydration.</li>
	<li>Depilate its left precordial chest and neck with depilatory cream, and use cotton swabs to remove hairs. Disinfect its skin with iodophor, followed by 70% alcohol to clean the area three times.</li>
	<li>Perform a midline cervical incision (0.8-1.0 cm) under a microscope. Separate the skin, muscle, and tissue surrounding the trachea, and then insert a 20 G cannula into the trachea. 
	NOTE: Observe while inserting the cannula into the trachea under microscopic view to ensure that the tube is not inserted into the esophagus.</li>
	<li>Connect the mouse to a rodent ventilator set at 0.2 mL tidal volume with 150 strokes per minute.</li>
	<li>Incise the mouse chest obliquely along the midclavicular line with a sterile scissor. Dissect the subcutaneous tissue to expose the ribs.</li>
	<li>Use sterile scissors to cut the skin obliquely along the left mid-clavicular line (0.8-1.0 cm). Perform left thoracotomy to separate the third and fourth ribs to expose the heart. Use a rib retractor for a clear view.</li>
	<li>Open the pericardium with curved forceps, and then ligate the left anterior descending artery (below the left atrium appendage) using a 7-0 silk suture. A pale anterior wall of the left ventricle indicates that the myocardial perfusion is successfully interrupted.</li>
	<li>Release the rib retractor, and stitch the thoracic incision in layers with a 5-0 silk suture while expelling air from the chest. Stitch the neck incision with a 5-0 suture silk suture.</li>
	<li>Remove the tracheal tube from the mouse once its spontaneous breathing is restored. Intraperitoneally inject 0.5 mL of pre-warmed sterile saline. Inject buprenorphine (0.1 mg/kg) every 12 h subcutaneously to minimize pain.</li>
	<li>Finally, place the mouse on the heating pad to wait for resuscitation. Monitor the mouse to ensure it does not suffer from excessive dyspnea or hemorrhaging via visual observation.</li>
	<li>Return the mouse to its cage after it wakes up. For the first week, monitor the mouse daily to verify that its mobility, grooming, and eating habits are adequate. According to the experiment&#39;s needs, harvest the heart at different time points after MI. 
	&#8203;NOTE: In this protocol, the single-cell suspension was prepared with the heart 2 weeks after MI.</li>
</ol>
2. Preparation of the cardiac single-cell suspension
<ol>
	<li>Preparation of the solutions and media
	<ol>
		<li>Cardiac tissue dissociation buffer: Mix 10 mg of collagenase IV (600 U/mL), 5 mg of dispase II (0.5 U/mL), and 50 &#181;L of DNase I (100 &#181;g/mL) in 5 mL of RPMI 1640. Pre-warm this medium to 37 &#176;C in a water bath before use.</li>
		<li>Cell wash buffer: Prepare 1% BSA or 10% FBS in phosphate-buffered saline (PBS, PH 7.4), and keep at 4 &#176;C or on ice.</li>
		<li>Perfusion solution: Pre-cool PBS (PH 7.4) as a perfusion solution.</li>
		<li>Red blood cell (RBC) lysis buffer: Use the RBC lysis buffer as mentioned by the manufacturer. 
		NOTE: Refer to the Table of Materials for the list of reagents and equipment used in this study.</li>
	</ol>
	</li>
	<li>Euthanize the mouse by an intraperitoneal pentobarbital sodium (150 mg/kg) injection.</li>
	<li>Heart dissection
	<ol>
		<li>Place the mouse in a supine position by fixing its hindlimbs in place with adhesive tape.</li>
		<li>Spray the body with 70% ethanol, and then cut vertically through the abdominal skin and muscles while avoiding piercing the liver. Carefully open the thorax while avoiding puncturing the heart.</li>
		<li>Use the pre-cooled PBS (PH 7.4) to perfuse the left ventricle until the colorless perfusion fluid is observed (approximately 15 mL of PBS [pH 7.4] is needed).</li>
		<li>Gently lift the heart from the thorax with forceps, and cut away the excess tissue attached to the outside of the heart with ophthalmic scissors. Place the heart in 1 mL of pre-cooled PBS (PH 7.4) in a 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Enzymatic dissociation of the heart tissue
	<ol>
		<li>Transfer the mouse heart into one well of a 24-well plate with 150 &#181;L of the cardiac tissue dissociation buffer placed on ice, and cut it into small pieces (~1 mm) with a scissor.</li>
		<li>Transfer the minced heart tissue into a 15 mL centrifuge tube with 5 mL of the tissue dissociation buffer. 
		NOTE: The recommended cardiac tissue dissociation buffer volume is 5 mL/heart.</li>
		<li>Place the centrifuge tube in the electrically thermostatic reciprocating shaker at 125 rpm for 1 h at 37 &#176;C. Pipet the tissue suspension up and down 10 times every 20 min.</li>
		<li>Filter the cell suspension through a 70 &#181;m cell strainer to remove undigested tissue and clumps. Add 25 mL of the cell wash buffer to rinse the original tube, and then transfer it to rinse the cell strainer. Next, filter the cell suspension through a 40 &#181;m cell strainer to further remove cell clumps.</li>
		<li>Centrifuge the filtered cell suspension at 300 x g for 5 min at 4 &#176;C. Discard the supernatant, and then resuspend the cell sample in 1x RBC lysis buffer for 5 min at room temperature (RT).</li>
		<li>Add four times more volume of the cell wash buffer, and centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant, and resuspend the cells in 10 mL of the cell wash buffer. Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant, and resuspend the cells in 1 mL of the cell wash buffer.</li>
		<li>Mix 18 &#181;L of the cell suspension with 2 &#181;L of acridine orange (AO)/propidium iodide (PI) staining solution (9:1), and add 10 &#181;L of the mixture to a counting chamber slide. Evaluate the cell numbers and viability using an automatic cell counter.</li>
	</ol>
	</li>
</ol>

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<li>Song, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</li>
<li>Jaitin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Massively+parallel+single-cell+RNA-seq+for+marker-free+decomposition+of+tissues+into+cell+types%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.</a> Science. 343 (6172), 776-779 (2014).</li>
<li>Massaia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+gene+expression+to+understand+the+dynamic+architecture+of+the+heart%5BTitle%5D)+AND+%22Frontiers+in+Cardiovascular+Medicine%22%5BJournal%5D)">Single cell gene expression to understand the dynamic architecture of the heart.</a> Frontiers in Cardiovascular Medicine. 5, 167(2018).</li>
<li>Papalexi, E., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA+sequencing+to+explore+immune+cell+heterogeneity%5BTitle%5D)+AND+%22Nature+Reviews.+Immunology%22%5BJournal%5D)">Single-cell RNA sequencing to explore immune cell heterogeneity.</a> Nature Reviews. Immunology. 18 (1), 35-45 (2018).</li>
<li>Jin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA+sequencing+reveals+the+temporal+diversity+and+dynamics+of+cardiac+immunity+after+myocardial+infarction%5BTitle%5D)+AND+%22Small+Methods%22%5BJournal%5D)">Single-cell RNA sequencing reveals the temporal diversity and dynamics of cardiac immunity after myocardial infarction.</a> Small Methods. 6 (3), 2100752(2022).</li>
<li>Tombor, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+sequencing+reveals+endothelial+plasticity+with+transient+mesenchymal+activation+after+myocardial+infarction%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.</a> Nature Communications. 12 (1), 681(2021).</li>
<li>Virag, J. A., Lust, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+artery+ligation+and+intramyocardial+injection+in+a+murine+model+of+infarction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Coronary artery ligation and intramyocardial injection in a murine model of infarction.</a> Journal of Visualized Experiments. (52), e2581(2011).</li>
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<li>Gladka, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+sequencing+of+the+healthy+and+diseased+heart+reveals+cytoskeleton-associated+protein+4+as+a+new+modulator+of+fibroblasts+activation%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Single-cell sequencing of the healthy and diseased heart reveals cytoskeleton-associated protein 4 as a new modulator of fibroblasts activation.</a> Circulation. 138 (2), 166-180 (2018).</li>
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The authors declare that they have no competing interests.

This article aimed to describe a protocol to isolate single non-cardiomyocytes from mouse hearts post MI. The protocol can be applied to isolate different types of cells in the post-MI microenvironment with high quality, including immune cells, endothelial cells, and fibroblasts. Three essential factors are crucial for obtaining a high-quality cell suspension for single-cell sequencing. The first one is the setting of the enzymatic digestion. It is important to control the time of digestion and the volume and concentration of the digestive enzymes to assure both cell viability and isolation efficiency. In this case, we appropriately increased the concentration of the enzyme and prolonged the digestion time. Here, we recommend a digestion duration of 45 min to 1 h. A shorter duration may increase the cell viability but may lead to insufficient cardiac tissue digestion and may increase the amount of cell clumps. In addition, we used DNase I to prevent cell aggregation because lytic cells can release free DNA to wrap the surrounding cells to form cell clumps13.&#160;The second essential factor is using a cell washing solution containing BSA or FBS. BSA or FBS can not only protect the activity of the enzymes but also improve the viability of the cells. The final essential factor is facilitating FACS to eliminate cell fragments and dead cells. Cell fragments can result in an inaccurate cell count and viability. These factors together enhance the viability and purity of the cell suspension, making it more compatible with single-cell sequencing.
This protocol firstly describes the cell suspension preparation for single-cell sequencing, and it also includes additional procedures to enhance the quality of the cell suspension at a relatively low cost. In comparison with other protocols used by previous studies, we avoid using digestive enzymes that can cause the aggregation of cells induced by free DNA, such as trypsin14. The removal of the cell debris is one of the major challenges for single-cell studies, and some studies use dead cell removal kits to improve cell viability before running the samples14,15. However, here, we suggest using FACS to enhance the sample purity due to its high effectiveness in reducing dead cells and debris.
The single-cell suspension obtained by this protocol can be further analyzed by flow analysis or primary cell culture (standard aseptic procedures)18. As the digestive enzymes are relatively mild, it is suitable to digest cardiac tissue taken in different stages of MI, as well as that from healthy hearts. However, this protocol has certain limitations. For example, prolonged digestion at 37 &#176;C inevitably promotes the expression of stress genes19,20. This limitation can be solved by using low-temperature active protease or reducing the digestion time followed by FACS. Also, this protocol does not include isolating cardiomyocytes, and, thus, it is not suitable for studies on local tissue responses.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, and its mortality increasing throughout the world1. MI is caused by an insufficient blood supply to the surrounding myocardium, which can be a result of a coronary artery blockage that occurs with atherosclerotic plaque rupture. Although percutaneous coronary intervention (PCI) has reduced the mortality rates of acute MI patients, the high prevalence of heart failure post MI remains a problem2. The key pathophysiology underlying post-MI heart failure is the body&#39;s compensatory response to cardiac injuries, which involves replacing the dead cardiac muscle with non-contractile fibrotic scars. These adaptive responses significantly rely on local inflammation mediated by the interactions between multiple cell types and cardiac tissue cells, and this inflammation is now considered as a potential therapeutic target to reduce fibrotic scar formation and, thus, protect against post-MI heart failure3,4. Interestingly, the microenvironment at the infarction site experiences a time-dependent transition in the infiltrating cell types and functions at different stages of MI1,5. Many studies have shown that non-cardiomyocytes (e.g., immune cells, fibroblasts, and endothelial cells) play central roles in post-MI inflammation and tissue repair5,6. In recent years, single-cell sequencing has been widely used as a powerful tool for elucidating the involvements and functions of non-cardiomyocytes in the post-MI microenviroment7,8. This provides insights into the pathophysiology of post-MI injury and repair and the development of potential therapies against post-MI heart failure.
High-throughput RNA sequencing (RNA-Seq) is a technique used to study entire transcriptomes in great detail using next-generation sequencing (NGS)7,8,9. Recently, the development of scRNA-Seq has revolutionized the biomedical research field. Compared with conventional bulk sequencing, scRNA-Seq analyses gene expression profiles and transcriptional heterogeneity at the single-cell level7,8. This technique significantly promotes the research of the cellular pathophysiology of MI9,10 by identifying different circulating cell types in the post-MI microenvironment and uncovering the interaction between cardiomyocytes and non-cardiomyocytes. These findings further contribute to uncovering novel therapeutic targets for post-MI heart failure. In general, the scRNA-seq-based post-MI experiment includes three main sections: (1) the establishment of post-MI animal model; (2) the preparation of the cell suspension; and (3) sample sequencing and data analysis. Noticeably, preparing the cell suspension is the most critical step in the preparation of the scRNA-Seq experiment because the quality of the cell suspension determines the accuracy of the results.
This protocol is designed to extract a non-cardiomyocyte cell suspension from the post-MI cardiac tissue; significantly, the specific details for maintaining cell viability and resolution are included. Meanwhile, the equipment used in this protocol, such as surgical kits for mice, rodent ventilators, and centrifuges, can be found in most animal experiment centers and biomedical laboratories, and, thus, the experiment cost of this protocol is relatively low. Furthermore, if considering time points and sites of infarction as variables, this protocol can be applied to simulate a wide range of clinical scenarios, especially for the post-MI complications.

<table><tbody><tr><td>Acridine Orange / Propidium Iodide Stain</td><td>Logos biosystems</td><td>F23001</td><td></td></tr><tr><td>Automated&amp;nbsp;Cell&amp;nbsp;Counter</td><td>Logos biosystems</td><td>L20001</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Servicebio</td><td>G5001</td><td>Cytoprotective effect</td></tr><tr><td>Cell Counting Slides</td><td>Logos biosystems</td><td>L12001</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Gibco</td><td>17104019</td><td>Digestive enzymes</td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693</td><td>Digestive enzymes</td></tr><tr><td>Dnase I</td><td>Roche</td><td>11284932001</td><td>Prevent cell clumping</td></tr><tr><td>Falcon&amp;nbsp;40&amp;mu;m Cell Strainer</td><td>Falcon</td><td>352340</td><td>Remove cell clumps</td></tr><tr><td>Falcon&amp;nbsp;70&amp;mu;m Cell Strainer</td><td>Falcon</td><td>352350</td><td>Remove undigested tissue and clumps</td></tr><tr><td>Flow Cell Sorter</td><td>Beckman Coulter</td><td>B25982</td><td></td></tr><tr><td>Iodophor</td><td>OU QING SI</td><td>10054963976859</td><td></td></tr><tr><td>Needle Holder</td><td>FST</td><td>12061-01</td><td></td></tr><tr><td>Ophthalmic Forceps</td><td>RWD</td><td>F14012-10</td><td></td></tr><tr><td>Ophthalmic Scissors</td><td>RWD</td><td>S11036-08</td><td></td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Servicebio</td><td>G4202-500ML</td><td></td></tr><tr><td>RBC Lysis Buffer</td><td>Beyotime</td><td>C3702-120ml</td><td>Remove red blood cells</td></tr><tr><td>Rib Retractor</td><td>FST</td><td>17005-04</td><td></td></tr><tr><td>Rodent Ventilator</td><td>Harvard</td><td>730043</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875093</td><td>Solvent&amp;nbsp;solution of enzyme</td></tr><tr><td>Sterile Scissor</td><td>RWD</td><td>S14014-10</td><td></td></tr></tbody></table>

preparation of a non-cardiomyocyte cell suspension for single-cell rna sequencing from a post-myocardial infarction adult mouse heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

A single-cell suspension with high vitality was obtained by implementing section 2 of this protocol. However, cell fragments could still be observed (Figure 1A); hence, fluorescence-activated cell sorting (FACS) was performed to further improve the quality16.After FACS, the average cell size reduces from 9.6 &#181;m to 9.1 &#181;m (Table 1), which suggests that the proportion of cell fragments can be effectively reduced in the cell suspension by FACS (Figure 1B). The gating strategy used for FACS is shown in Figure 1C.
In addition to the images in Figure 1, the cell concentration, average cell size, and cell viability were measured by a cell counter (Table 1). The differences in cell concentrations were due to the volume of the cell suspension. Noticeably, the average cell size decreased after FACS (Table 1), which shows that FACS can effectively reduce cell clumping and remove cell fragments.
ScRNA-seq was performed to study the cardiac immunological microenvironment while verifying the cell suspension quality obtained through this protocol. After preparing the single-cell suspension, we firstly sorted the cells that expressed surface protein CD45, which is a common marker for a wide range of immune cells. Next, we performed scRNA-seq on CD45+ cells using the 10X Genomics platform17. In this way, we pooled 14,217 individual cells from three healthy heart samples into a merged data set after quality control (Table 2). The unsupervised clustering and reduction in t-distributed stochastic neighbor embedding (t-SNE) dimensionality showed an obvious separation of seven types of immune cells (Figure 2). Moreover, each immune cell type showed a high expression of classic markers. Conclusively, these results show that the single-cell suspension prepared by this protocol has a high potency for single-cell sequencing.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64290/64290fig01.jpg" /> 
Figure 1: Cell viability in the cardiac single-cell suspension. (A)&#160;Cell viability without FACS. The black arrowheads indicate cell fragments (no fluorescence). (B) Cell viability with FACS. The yellow arrow indicates live cells (green fluorescence), and the red arrow indicates dead cells (red fluorescence).(C) Gating strategy. Scale bar: 50 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64290/64290fig01large.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/64290/64290fig02v3.jpg" /> 
Figure 2: Single-cell RNA sequencing of cardiac CD45+ cells from three mouse heart samples. (A)&#160;tSNE plot of CD45+ cells showing the immune cell populations identified based on canonical marker genes. (B) Heatmap of the top five differentially expressed genes in each subpopulation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64290/64290fig02largev3.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></td>
			<td>Before FACS</td>
			<td>After FACS</td>
		</tr>
		<tr>
			<td>Total cell concentration (cells/mL)</td>
			<td>1.51 x 106&#160;</td>
			<td>3.40 x 105</td>
		</tr>
		<tr>
			<td>Live cell concentration (cells/mL)</td>
			<td>1.40 x 106&#160;</td>
			<td>3.15 x 105</td>
		</tr>
		<tr>
			<td>Dead cell concentration (cells/mL)</td>
			<td>1.09 x 105</td>
			<td>2.48 x 104</td>
		</tr>
		<tr>
			<td>Viability (%)</td>
			<td>92.8</td>
			<td>92.7</td>
		</tr>
		<tr>
			<td>Average cell size (&#956;m)</td>
			<td>9.6</td>
			<td>9.1</td>
		</tr>
	</tbody>
</table>
Table 1: Cardiac single-cell suspension before and after FACS. The cell densities of the cardiac single-cell suspension obtained before and after FACS are compared using an automated cell counter.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Estimates</td>
		</tr>
		<tr>
			<td>Estimated Number of Cells</td>
			<td>14,217</td>
		</tr>
		<tr>
			<td>Mean Reads per Cell</td>
			<td>81,017</td>
		</tr>
		<tr>
			<td>Median Genes per Cell</td>
			<td>1,374</td>
		</tr>
		<tr>
			<td>Total Genes Detected</td>
			<td>18,424</td>
		</tr>
		<tr>
			<td>Median UMI Counts per Cell</td>
			<td>4,021</td>
		</tr>
		<tr>
			<td>Valid Barcodes</td>
			<td>98.4%</td>
		</tr>
		<tr>
			<td>Reads Mapped to Genome</td>
			<td>95.1%</td>
		</tr>
	</tbody>
</table>
Table 2: Quality control statistics. The table lists the different estimates of the single-cell data of healthy mouse hearts.

Watch this Scientific Journal Video about Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart at JoVE.com

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

preparation-non-cardiomyocyte-cell-suspension-for-single-cell-rna

Universidad San Sebastian

Research

JoVE Journal

Medicine

2.0K Views.  Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province. Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Video: Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Developmental Biology

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Biology

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 ...

Immunology and Infection

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac 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. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

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Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

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

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice