Name: flow cytometry-based quantification and analysis of myocardial b-cells
Uploaded: 2022-08-17T14:50:00.000+00:00
Duration: 12 min 46 s
Description: Watch this Scientific Journal Video about Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells at JoVE.com

This study was funded by NHLBI grants 5K08HLO145108-03 and 1R01HL160716-01 awarded to Luigi Adamo.
The Aurora Flow Cytometer used to develop this study was funded by NIH Grant S10OD026859. We acknowledge the support of the JHU Ross Flow Cytometry Core.

All experiments described in this manuscript were performed with the approval of the IACUC at Johns Hopkins University School of Medicine.
1. Preparations
<ol>
	<li>Prepare the FACS Buffer, as described in Table 1.</li>
	<li>Ensure there is enough CO2 to euthanize the animals.</li>
	<li>Prepare the dissection space (place the bench pad and place the tape and dissection tools nearby).</li>
	<li>Label the 15 mL tubes, put 3 mL of HBSS with calcium and magnesium in each tube (see Table of Materials) and place them on ice (one tube per heart and an extra one for the Reference group/flow cytometry controls). Also, fill the small (35 mm) Petri dishes with HBSS.</li>
	<li>Turn on the temperature-controlled shaker, set it to 37 &#176;C, and pre-chill the centrifuge at a temperature of 4 &#176;C.</li>
	<li>Prepare the syringe pump and set the flow rate to 3 mL/min. Fill a 20 mL syringe with HBSS, prime the tube line with buffer, and remove any bubbles.</li>
</ol>
2. Intravascular B-cell staining (in vivo)
<ol>
	<li>Weigh a 12-week-old male mouse of the C57BL/6J strain. Load a 3/10 cc insulin syringe with 100 &#181;L of the B220 antibody dilution (1:10, in PBS). Cover the syringe with aluminum foil to protect from light.</li>
	<li>Anesthetize one mouse.
	<ol>
		<li>Apply an intraperitoneal injection of 2% 2,2,2-tribromoethanol with a dose of 400 mg/kg and wait 10-20 min. 
		NOTE: Monitor the mouse&#39;s breathing and movement. Once the mouse stops moving, stimulate pain by pinching the toe; the absence of a withdrawal reflex indicates adequate anesthesia.</li>
		<li>If adequate anesthesia is not achieved within the course of 20 min, an additional dose of anesthetic at 100 mg/kg can be administered, and an assessment of mouse kinetics, withdrawal reflex, and respiration should be made every 5 min.</li>
	</ol>
	</li>
	<li>Place the mouse on the lab bench on a bench pad inclined toward one side, gently pull down the skin of the back, and slightly press the body against the bench to make the eye protrude.</li>
	<li>Inject the diluted B220 antibody from step 2.1 by retroorbital injection.
	<ol>
		<li>Introduce the syringe into the eye at 45&#176; from the sagittal plane of the mouse&#39;s head. Slowly inject the solution. If resistance is felt, remove the syringe and enter again. Wait for 2 min. 
		NOTE: Prolonged incubation time would jeopardize the ability to discriminate intra-vascular vs extra-vascular B cells as it would give time to the injected antibody to diffuse outside of the vasculature.&#160;</li>
	</ol>
	</li>
	<li>Euthanize the mouse as per the IACUC regulations.
	<ol>
		<li>Open the CO2 flow to the euthanasia chamber at a rate of 0.5 L/min, or the recommended flow rate based on the size of your chamber.</li>
		<li>Place the mouse in the chamber for the recommended time, ensuring it is no longer breathing. Remove it and perform cervical dislocation as a secondary method of euthanasia.</li>
		<li>Once the mouse is euthanized, promptly proceed to organ harvesting and perfusion.</li>
	</ol>
	</li>
</ol>
3. Heart collection
<ol>
	<li>Chest opening.
	<ol>
		<li>Hold the skin of the mouse with forceps at the epigastric area. Make a 2 mm opening in the skin using scissors and use the forceps to peel the skin away to reveal the fascia layer, a shiny translucent layer, of the chest and abdomen. Cut at the epigastrium through the fascia and peritoneum to open the abdominal wall and reveal the xiphoid process. Clamp the xiphoid process using hemostatic forceps.</li>
		<li>With the scissors, make a vertical cut through the chest wall at the level of the mid-clavicular line on each side and lift the anterior chest wall to reveal the mediastinum content, using hemostatic forceps as a weight to maintain the opening.</li>
	</ol>
	</li>
	<li>Heart perfusion and extraction.
	<ol>
		<li>Using forceps, remove any fat or thymic tissue surrounding the heart.</li>
		<li>Hold the aorta securely from behind using forceps. Introduce the syringe needle (25 G) into the apex of the heart. Perfuse with 3 mL of HBSS at a rate of 3 mL/min. 
		NOTE: Monitor the perfusion. The liver should fill, and blood in the coronary vessels should be entirely removed.&#160;Use of a syringe pump calibrated to 1 mL/ minute is recommended to maintain a steady flow.</li>
		<li>Cut the aorta using scissors and take out the heart. Wash the heart in the Petri dish with HBSS to remove any remaining blood. Using scissors and forceps, remove the fat, connective tissue and thymus, and dry the heart. Weigh the isolated heart and note the weight in milligrams. Mince the heart at room temperature into small pieces (1-2 mm) with a blade. 
		NOTE: Adult mouse hearts may weigh between 0.090-0.210 mg.</li>
		<li>Measure the mass of heart tissue and place 60 mg of the minced heart to be digested in a 15 mL tube with 3 mL of HBSS. Retain the remaining heart tissue and place in a tube labelled &#34;Reference control tissue&#34;; this will be used for compensation control for live-dead signal and autofluorescence.</li>
	</ol>
	</li>
</ol>
4. Heart digestion and cell staining
<ol>
	<li>Add enzymes to each tube containing minced tissue (see Table 1). Add 0.5 &#181;L/mg of DNase 1 (300 Units for 60 mg of heart tissue), 0.5 &#181;L/mg of collagenase II (625 Units for 60 mg of heart tissue), and 0.2 &#181;L/mg of hyaluronidase (50 Units for 60 mg of heart tissue) to the tube.</li>
	<li>Place the digestion reaction in the shaker for 30 min at 300 rpm. Adjust the shaker rack to about 25&#176; of incline.</li>
	<li>Add 7 mL of HBSS to each of the 15 mL reaction tubes and centrifuge at 250 x g for 5 min at 4 &#176;C with acceleration and breaking set to moderate or slow. Decant the supernatant and resuspend each pellet in 5 mL of ACK lysis buffer to remove red blood cells. Incubate in the ACK lysis buffer for 5 min at room temperature.</li>
	<li>Add 10 mL of PBS to each tube, mix, and then filter into a 50 mL tube with a 40 &#181;m strainer. Ensure that all the debris is inside the filter chamber. Smash any debris on the filter with a syringe plunger until it is completely suspended within the filter chamber.</li>
	<li>Add PBS through the filter until the volume reaches 25 mL per heart; this will allow the suspended cells to pass through the filter. Add an additional 25 mL of PBS to the Reference control tissue tube and divide the volume into different tubes (25 mL each). Label one of the tubes &#34;Unstained&#34; and the other one as &#34;Live-dead&#34;. Centrifuge at 250 x g for 5 min at 4 &#176;C. 
	NOTE: At this point, prepare the dilution for the live-dead stain (dilution: 1:500 in 1x PBS).</li>
	<li>Decant the supernatant, resuspend the pellet in the Unstained tube in 100 &#181;L of PBS and each of the other pellets in 100 &#181;L of the live-dead stain dilution. Incubate on ice for 30 min in the dark, covering the ice bucket with aluminum foil.</li>
	<li>Add 500 &#181;L of FACS buffer to each sample and transfer it into a labelled FACS tube through a 40 &#181;m filter. Centrifuge the FACS tubes at 250 x g for 5 min at 4 &#176;C. Discard the supernatant. Resuspend the pellet in 500 &#181;L of FACS buffer.</li>
	<li>Add 50 &#181;L of Fc blocking solution (see Table 1) to each tube, except for the Unstained and Live-dead tubes. Mix and incubate for 5 min.</li>
	<li>Add 50 &#181;L of antibody mix solution (see Table 1) to each tube, except for the Unstained and Live-dead tubes, and incubate for 30 min in the dark, covering the ice bucket with aluminum foil.</li>
	<li>Add 2 mL of FACS buffer to each tube and centrifuge at 250 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend in 300-500 &#181;L of FACS buffer.</li>
</ol>
5. Flow cytometry
<ol>
	<li>Setup instrument control settings. 
	NOTE:&#160;In this protocol the sample is analyzed with a 4 lasers Cytek Aurora flow cytometer. Another appropriate flow cytometer could be used to detect the markers of interest.&#160;
	<ol>
		<li>Open the instrument control software (see Table of Materials). At the top of the window, select the Acquisition tab and click on New +. A window called Create New Experiment will display.</li>
		<li>In the Library section, in the field Type to filter, type PE, PerCP-Cy5.5, BV421, Alexa Fluor 700, and Zombie Aqua, clicking Add after typing each. The fluorophore names should be seen to the right. Then click Next to proceed to the Group tab.</li>
		<li>In the Group tab, click the symbol with a tube to add the heart for the analysis.</li>
		<li>Click on the + Reference group and a window will display. In the fluorescent tag column select Zombie Aqua, and in the control type column select Cells, then click Save.</li>
		<li>Click Next &#62; Markers tab; a table will display with the names of the fluorophores. Type the corresponding cell marker in the first row of each fluorophore column and type Enter: CD45 for PerCP-Cy5.5; CD19 for BV421; CD11b for Alexa Fluor 700; B220 for PE; and live-dead for Zombie Aqua.</li>
		<li>Click Next twice to go to the Acquisition tab. In Events to Record of the experiment group type 10,000,000, and in the same field for the reference group line type 200,000. Click Save and Open. The other settings should remain as default, Stopping Time to 10,000, and Stopping Volume to 3,000.</li>
		<li>On the bottom left click on Instrument control. Set Voltage to FSC-50, SSC-175, SSC-B-175. Then, select the Acquisition Control. For the Flow rate option, select High from the drop-down menu.</li>
	</ol>
	</li>
	<li>Acquire reference samples with a cytometer and unmix in software.
	<ol>
		<li>Vortex the Unstained heart tube and place it securely on the sample injection port (SIP). Ensure the green indicator arrow is pointing to the correct sample, then click Start in the Acquisition Control panel of the cytometer software and wait until the events are recorded. Do the same for the Live-dead-stained heart tissue.</li>
		<li>Select the Unmix menu and set the following controls:
		<ol>
			<li>Select the checkboxes for PE, PerCP-Cy5.5, BV421, and Alexa Fluor 700 in the column from library. Ensure Zombie Aqua is unchecked in the same column. At the bottom, check the option of Autofluorescence as a Fluorescent Tag.</li>
			<li>Click Next to proceed to the Identify Positive/Negative Populations tab. In this tab, a plot of FSC-A vs. SSC-B-A will be displayed. Click and drag the points of the polygon to select an area between 1.0 M and 4.0 M on the FSC-A axis, and between 0.2 M and 3.0 M on the SSC-B-A axis.</li>
			<li>In the next plot to the right, V5-A will be displayed on the X-axis. Move the gates to select half of the peak on the far right as positive and a region on the left side of the plot as negative. In the plot titled Reference Group - Unstained, select a population in a similar range. For this the unstained no positive-negative has to be set.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Acquire experimental samples.
	<ol>
		<li>For each tube containing a sample for an individual mouse, vortex the tube and place it securely on the SIP. Ensure the green indicator arrow is pointing to the correct sample, then click Start in the Acquisition Control panel of the cytometer software, and wait until the events are recorded.</li>
	</ol>
	</li>
	<li>Gating strategy.
	<ol>
		<li>Select the Default Unmixed Worksheet tab. Create an FSC-A vs. SSC-A plot using the Dot plot tool on top. Select events higher than 0.4 M on the FSC-A axis and higher than 0.8 M on the SSC-A axis using the Polygon selection tool. Double click in this selection and a new plot will display.</li>
		<li>From the newly created plot, right-click on the X axis label and select AF-A; right-click on the Y axis label and select Live-dead stain. Click on the Rectangle selection tool on top and select all the events below 105 on the live-dead axis&#160;that correspond to the lower live-dead signal. Double click in this selection and a new plot will display.</li>
		<li>In the new plot, right-click to select PerCP-Cy5.5-A for the X axis and SSC-A for the Y axis. Using the Rectangle selection tool, select the events higher than 104 on the PerCP-Cy5.5-A axis that correspond to the CD45 positive cells. Double click on the selection and a new plot will display.</li>
		<li>On the new plot, right-click to select FSC-A for the X axis and FSC-H for the Y axis. Using the Polygon selection tool&#160;select the events that follow a trend on which FSC-A value and SSC-A value are the same; with this the cell doublets will be removed and the population in the selection will be referred to as the CD45+ population. Double click on the selection to display a new plot with the CD45+ population.</li>
		<li>From the CD45+ population plot, right-click to select BV421 on the X axis vs. SSC-A on the Y axis. Using the Polygon selection tool, select the population to the right on the BV421 axis. This is the B-cell population. Double click twice in this population to create two new plots with the B-cell population.
		<ol>
			<li>Right-click to setup the first B-cell plot with PE-A at the X axis and BV421-A on the Y axis. The population to the right corresponds to the intravascular B-cells, and the population to the left represents the interstitial B-cells. Use the Polygon selection tool to make a selection of both.</li>
			<li>Right-click to setup the second B-cell plot with Alexa Fluor 700-A on the X axis and SSC-A on the Y axis. The population at the left corresponds to the CD11b- cells, and the events at the right (if any) correspond to the CD11b+ cells.</li>
			<li>Right click on each selection and then click Gate Properties. Click on Count and % Parent to display the sizes and percentages. Record the number of events and percentages for further data analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Adamo, L., Rocha-Resende, C., Mann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+B+lymphocytes+in+cardiovascular+disease.">The emerging role of B lymphocytes in cardiovascular disease.</a> Annual Review of Immunology. 38, 99-121 (2020).</li><li>Gowans, J. L., Knight, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+route+of+re-circulation+of+lymphocytes+in+the+rat.">The route of re-circulation of lymphocytes in the rat.</a> Proceedings of the Royal Society of London. Series B: Biological Sciences. 159 (975), 257-282 (1964).</li><li>Kunkel, E. J., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+the+tissue-specific+migration+of+lymphocytes.">Chemokines and the tissue-specific migration of lymphocytes.</a> Immunity. 16 (1), 1-4 (2002).</li><li>Tanaka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinants+controlling+homeostatic+recirculation+and+tissue-specific+trafficking+of+lymphocytes.">Molecular determinants controlling homeostatic recirculation and tissue-specific trafficking of lymphocytes.</a> International Archives of Allergy and Immunology. 134 (2), 120-134 (2004).</li><li>Rocha-Resende, 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=Developmental+changes+in+myocardial+B+cells+mirror+changes+in+B+cells+associated+with+different+organs.">Developmental changes in myocardial B cells mirror changes in B cells associated with different organs.</a> JCI Insight. 5 (16), (2020).</li><li>Adamo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+B+cells+are+a+subset+of+circulating+lymphocytes+with+delayed+transit+through+the+heart.">Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart.</a> JCI Insight. 5 (3), 139377 (2020).</li><li>Adamo, L., 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=Modulation+of+subsets+of+cardiac+B+lymphocytes+improves+cardiac+function+after+acute+injury.">Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury.</a> JCI Insight. 3 (11), (2018).</li><li>Rocha-Resende, C., Pani, F., Adamo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+modulate+the+expression+of+MHC-II+on+cardiac+CCR2(-)+macrophages.">B cells modulate the expression of MHC-II on cardiac CCR2(-) macrophages.</a> Journal of Molecular and Cellular Cardiology. 157, 98-103 (2021).</li><li>Zouggari, Y., 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=B+lymphocytes+trigger+monocyte+mobilization+and+impair+heart+function+after+acute+myocardial+infarction.">B lymphocytes trigger monocyte mobilization and impair heart function after acute myocardial infarction.</a> Nature Medicine. 19 (10), 1273-1280 (2013).</li><li>Wu, L., 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=IL-10-producing+B+cells+are+enriched+in+murine+pericardial+adipose+tissues+and+ameliorate+the+outcome+of+acute+myocardial+infarction.">IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction.</a> Proceedings of the National Academy of Sciences. 116 (43), 21673-21684 (2019).</li><li>Heinrichs, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+healing+myocardium+mobilizes+a+distinct+B-cell+subset+through+a+CXCL13-CXCR5-dependent+mechanism.">The healing myocardium mobilizes a distinct B-cell subset through a CXCL13-CXCR5-dependent mechanism.</a> Cardiovascular Research. 117 (13), 2664-2676 (2021).</li><li>Sun, Y., 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=Splenic+marginal+zone+B+lymphocytes+regulate+cardiac+remodeling+after+acute+myocardial+infarction+in+mice.">Splenic marginal zone B lymphocytes regulate cardiac remodeling after acute myocardial infarction in mice.</a> Journal of the American College of Cardiology. 79 (7), 632-647 (2022).</li><li>Yan, 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=Temporal+dynamics+of+cardiac+immune+cell+accumulation+following+acute+myocardial+infarction.">Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.</a> Journal of Molecular and Cellular Cardiology. 62, 24-35 (2013).</li><li>Iwata, 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=Autoimmunity+against+the+second+extracellular+loop+of+beta(1)-adrenergic+receptors+induces+beta-adrenergic+receptor+desensitization+and+myocardial+hypertrophy+in+vivo.">Autoimmunity against the second extracellular loop of beta(1)-adrenergic receptors induces beta-adrenergic receptor desensitization and myocardial hypertrophy in vivo.</a> Circulation Research. 88 (1), 578-586 (2001).</li><li>Jahns, 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=Direct+evidence+for+a+beta+1-adrenergic+receptor-directed+autoimmune+attack+as+a+cause+of+idiopathic+dilated+cardiomyopathy.">Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy.</a> The Journal of Clinical Investigation. 113 (10), 1419-1429 (2004).</li><li>Christ, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+against+the+beta1+adrenoceptor+from+patients+with+dilated+cardiomyopathy+prolong+action+potential+duration+and+enhance+contractility+in+isolated+cardiomyocytes.">Autoantibodies against the beta1 adrenoceptor from patients with dilated cardiomyopathy prolong action potential duration and enhance contractility in isolated cardiomyocytes.</a> Journal of Molecular and Cellular Cardiology. 33 (8), 1515-1525 (2001).</li><li>Jane-wit, D., 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=Adrenergic+receptor+autoantibodies+mediate+dilated+cardiomyopathy+by+agonistically+inducing+cardiomyocyte+apoptosis.">Adrenergic receptor autoantibodies mediate dilated cardiomyopathy by agonistically inducing cardiomyocyte apoptosis.</a> Circulation. 116 (4), 399-410 (2007).</li><li>Ludwig, R. 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=Mechanisms+of+autoantibody-induced+pathology.">Mechanisms of autoantibody-induced pathology.</a> Frontiers in Immunology. 8, 603 (2017).</li><li>Haudek, S. B., 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=Fc+receptor+engagement+mediates+differentiation+of+cardiac+fibroblast+precursor+cells.">Fc receptor engagement mediates differentiation of cardiac fibroblast precursor cells.</a> Proceedings of the National Academy of Sciences. 105 (29), 10179-10184 (2008).</li><li>Staudt, A., Eichler, P., Trimpert, C., Felix, S. B., Greinacher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc(gamma)+receptors+IIa+on+cardiomyocytes+and+their+potential+functional+relevance+in+dilated+cardiomyopathy.">Fc(gamma) receptors IIa on cardiomyocytes and their potential functional relevance in dilated cardiomyopathy.</a> Journal of the American College of Cardiology. 49 (16), 1684-1692 (2007).</li><li>Zhang, 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+role+of+natural+IgM+in+myocardial+ischemia-reperfusion+injury.">The role of natural IgM in myocardial ischemia-reperfusion injury.</a> Journal of Molecular and Cellular Cardiology. 41 (1), 62-67 (2006).</li><li>Zhang, 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=Identification+of+a+specific+self-reactive+IgM+antibody+that+initiates+intestinal+ischemia/reperfusion+injury.">Identification of a specific self-reactive IgM antibody that initiates intestinal ischemia/reperfusion injury.</a> Proceedings of the National Academy of Sciences. 101 (11), 3886-3891 (2004).</li><li>Schulze, K., Becker, B. F., Schauer, R., Schultheiss, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+ADP-ATP+carrier--an+autoantigen+in+myocarditis+and+dilated+cardiomyopathy--impair+cardiac+function.">Antibodies to ADP-ATP carrier--an autoantigen in myocarditis and dilated cardiomyopathy--impair cardiac function.</a> Circulation. 81 (3), 959-969 (1990).</li><li>Matsumoto, Y., Park, I. K., Kohyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+epitope+spreading+is+a+critical+step+for+the+switch+from+C-protein-induced+myocarditis+to+dilated+cardiomyopathy.">B-cell epitope spreading is a critical step for the switch from C-protein-induced myocarditis to dilated cardiomyopathy.</a> The American Journal of Pathology. 170 (1), 43-51 (2007).</li><li>Caforio, A. L. P., 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=Current+state+of+knowledge+on+aetiology,+diagnosis,+management,+and+therapy+of+myocarditis:+a+position+statement+of+the+European+Society+of+Cardiology+Working+Group+on+Myocardial+and+Pericardial+Diseases.">Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases.</a> European Heart Journal. 34 (33), 2636-2648 (2013).</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>Yu, Y. 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=A+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues.">A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues.</a> PLoS One. 11 (3), 0150606 (2016).</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>Horckmans, 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=Pericardial+adipose+tissue+regulates+granulopoiesis,+fibrosis+and+cardiac+function+after+myocardial+infarction.">Pericardial adipose tissue regulates granulopoiesis, fibrosis and cardiac function after myocardial infarction.</a> Circulation. 137 (9), 948-960 (2017).</li><li>Lavine, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences. 111 (45), 16029-16034 (2014).</li><li>Bajpai, G., Lavine, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+macrophage+subsets+and+stromal+cells+from+human+and+mouse+myocardial+specimens.">Isolation of macrophage subsets and stromal cells from human and mouse myocardial specimens.</a> Journal of Visualized Experiments. (154), e60015 (2019).</li><li>Anderson, K. 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=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nature Protocols. 9 (1), 209-222 (2014).</li><li>Coffman, R. L., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B220:+a+B+cell-specific+member+of+th+T200+glycoprotein+family.">B220: a B cell-specific member of th T200 glycoprotein family.</a> Nature. 289 (5799), 681-683 (1981).</li><li>Montecino-Rodriguez, E., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+B+cell+development+in+the+fetus+and+adult.">B-1 B cell development in the fetus and adult.</a> Immunity. 36 (1), 13-21 (2012).</li><li>Bermea, K., Bhalodia, A., Huff, A., Rousseau, S., Adamo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+B+cells+in+cardiomyopathy+and+heart+failure.">The role of B cells in cardiomyopathy and heart failure.</a> Current Cardiology Reports. , 01722-01724 (2022).</li><li>Zhao, T. 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=Rituximab+in+patients+with+acute+ST-elevation+myocardial+infarction:+an+experimental+medicine+safety+study.">Rituximab in patients with acute ST-elevation myocardial infarction: an experimental medicine safety study.</a> Cardiovascular Research. 118 (3), 872-882 (2022).</li><li>Kushnir, N., 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=B2+but+not+B1+cells+can+contribute+to+CD4++T-cell-mediated+clearance+of+rotavirus+in+SCID+mice.">B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice.</a> Journal of Virology. 75 (12), 5482-5490 (2001).</li></ol>

Luigi Adamo is co-founder of i-Cordis, LLC, a company focused on the development of immunomodulatory molecules for the treatment of heart failure, with a specific interest in derivatives of the B-cell modulating drug pirfenidone. The remaining authors have no conflicts to declare.

A growing body of evidence indicates that B-cells play an important role in the context of myocardial physiology and myocardial remodeling/adaptation to injury7,8,9,10,11,12,13,36. Flow cytometry is an excellent tool to study immune cell populations in any ...

B-lymphocytes are highly specialized immune cells that play an important role in both adaptive and innate immune responses1. There are two main populations of B-cells: a smaller population of B1 cells that are mostly produced during embryonic life, and a preponderant population of B2 cells that are produced in adult life in the bone marrow1. After maturing in the bone marrow, B-cells migrate to primary and secondary lymphoid organs. From there they continuously recirculate between lymphoid organs traveling through blood vessels and lymphatic vessels2. B-cells express specific antibodies on their surface, which function as receptors. When B-cells encounter an antigen that binds to their receptor, an activating signal can be triggered. Activated B-cells either migrate to the tissue where the antigen was found or go back to the bone marrow where they can mature into antibody-producing plasma cells3,4.
Recently, it has been appreciated that the heart harbors a sizeable population of B-cells. Studies in rodents have shown that B-cells colonize the heart early during embryonic development5, and that myocardial-associated B-cells are mostly intravascular, naive B2 cells adhered to the endothelium6,7, with a small percentage of B1 cells7. There are still many areas of uncertainty, but the available data indicates that B-cells play an important role both in the naive heart and in the context of myocardial adaptation to injury.
Studies in the naive murine heart have shown that at baseline myocardial B-cells are mostly located in the intravascular space, adhered to the endothelium (&#62;95% of murine cardiac B-cells were found to be located in the intravascular space). These B cells were found to have gene expression patterns different from those of circulating B cells isolated from the peripheral blood. Analysis of naive hearts from B-cell-deficient animals and syngeneic controls found that animals lacking B-cells had smaller hearts and higher ejection fraction6. All this evidence suggests that B-cells might modulate myocardial growth and/or myocardial function, and that not only interstitial but also intravascular B-cells could be responsible for such observations. B-cells were also found to modulate the phenotype of myocardial resident macrophages8.
Several studies have shown that B-cells play an important role in the context of myocardial adaptation to injury8,9,10,11,12,13. B-cells accumulate transiently in the injured heart, likely through a CXCL13-CXCR5 dependent mechanism11,13. From there, B-cells promote adverse cardiac remodeling through several mechanisms that include cytokine-mediated monocyte recruiting9,12. In addition, B-cells can produce antibodies against cardiac proteins that can promote the extension of cardiac damage and adverse cardiac remodeling through several mechanisms14,15,16,17,18,19,20,21,22,23,24,25. B-cells can also exert protective effects on the injured heart through the secretion of IL-1010.
As the number of groups investigating the role of B-cells in the naive and injured heart grows, it is becoming more and more important to define shared protocols to properly quantify and assess myocardial B-cells and thus avoid inconsistencies that have already started to appear in the literature. So far B-cells have in fact both been reported to be one of the most prevalent immune cells in the rodent heart7 and to be present at a markedly lower prevalence than myeloid cells26,27, or to be quite rare28. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury7,9,13, but one group reported no changes in the number of B-cells of the injured myocardium29. Studies on cardiac immune cells rarely give details on perfusion conditions and there is no consensus on the digestion conditions. Since in the rodent heart a large proportion of B-cells are intravascular and extraction of immune cells from the myocardium is highly dependent on the digestion method used, the differences reported in the literature might be the result of differences in organ perfusion and tissue digestion.
Presented here is a detailed method for flow cytometry-based quantification of murine myocardial B-cells that maximizes the yield of B-cell recovery by optimizing perfusion and digestion conditions and allows the discrimination of intravascular vs. extravascular myocardial B-cells6. This protocol is an adaptation and optimization of other similar protocols that distinguish between intravascular and interstitial immune cells28,30,31.
In this protocol, we standardize myocardial perfusion to eliminate B-cells floating&#160;in the intravascular space without removing biologically relevant B-cells adhered to the microvascular endothelium. Moreover, building on prior protocols that have described the use of intravenous injection of antibodies to distinguish intravascular from interstitial immune cells32, and taking advantage of the fact that B-cells express the surface marker B22033, we demonstrate how to distinguish intravascular vs. extravascular myocardial B-cells through intravascular injection of a B220-specific antibody immediately before the animal sacrifice and cardiac perfusion. This protocol is relevant to the research of any scientist interested in including the analysis of myocardial B-cells in the naive and injured heart. The widespread implementation of this protocol will reduce inconsistencies between research groups, will allow the analysis of changes in the intravascular and extravascular myocardial B-cell pools, and thus will bolster the advancement of discoveries in the field of cardiac immunology.
In summary, the protocol represents an optimized workflow to quantify and analyze myocardial B-cells via flow cytometry, and at the same time distinguish between cells located in the extravascular space and the intravascular space.

<table><tbody><tr><td>Alexa Fluor 700 anti-mouse/human CD11b Antibody</td><td>101222</td><td>BioLegend</td><td>100 &amp;micro;g 200 &amp;micro;L</td></tr><tr><td>(CellTreat 29481) Cell Strainer, 40 &amp;micro;m, Blue</td><td>QBIAP303</td><td>Southern Labware</td><td></td></tr><tr><td>0.5 mL Natural Microcentrifuge Tube</td><td>1605-0000</td><td>SealRite, USA Scientific</td><td></td></tr><tr><td>0.9% Sodium Chloride Injection, USP</td><td>114-055-101</td><td>Quality Biological</td><td>0.90%</td></tr><tr><td>1.5 mL Natural Microcentrifuge Tube</td><td>1615-5500</td><td>SealRite, USA Scientific</td><td></td></tr><tr><td>10 &amp;micro;L Graduated TipOne Filter Tips</td><td>11213810</td><td>USA Scientific</td><td></td></tr><tr><td>1000 &amp;micro;L Graduated TipOne Filter Tips</td><td>11267810</td><td>USA Scientific</td><td></td></tr><tr><td>15 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free</td><td>430052</td><td>Corning</td><td></td></tr><tr><td>1-Way Stop Valve, Polycarbonate</td><td>SVPT951</td><td>ECT Manufacturing</td><td></td></tr><tr><td>2,2,2-Tribromoethanol</td><td>T48402</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>200 &amp;micro;L Graduated TipOne Filter Tips</td><td>11208810</td><td>USA Scientific</td><td></td></tr><tr><td>3-Way Stop Valve, Polycarbonate</td><td>SVPT953</td><td>ECT Manufacturing</td><td></td></tr><tr><td>5 mL Polystyrene Round-Bottom Tube, 12 x 75 mm style</td><td>352054</td><td>Falcon, a Corning Brand</td><td></td></tr><tr><td>50 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free</td><td>430290</td><td>Corning</td><td></td></tr><tr><td>ACK (Ammonium-Chloride-Potassium) Lysing Buffer</td><td>118-156-101</td><td>Quality Biological</td><td>Osmolality: 290 + or -5% mOsm/Kg H20</td></tr><tr><td>Adapter 4x50ml, for 250 mL rectangular bucket in Rotor A-4-63</td><td>5810759005</td><td>Eppendorf</td><td></td></tr><tr><td>Adapter for 15 mL Centrifuge Tubes, 9 Tubes per Adapter, Conical Bottom for use with Rotor Model A-4-62</td><td>22638289</td><td>Eppendorf</td><td></td></tr><tr><td>Adapter for 15 round-bottom tubes 2.6 &amp;ndash; 7 mL, for 250 mL rectangular bucket in Rotor A-4-62</td><td>22638246</td><td>Eppendorf</td><td></td></tr><tr><td>Aluminum Foil 12 in x 75&#039; Roll .0007</td><td>UPC 109153</td><td>Reynolds Wrap</td><td></td></tr><tr><td>Anesthesia Induction Chamber - Mouse</td><td>RWD-AICMV-100</td><td>Conduct Science</td><td></td></tr><tr><td>BD Luer Slip Tip Syringe with attached needle 25 G x 5/8 in., sterile, single use, 1 mL</td><td>309626</td><td>BD Becton, Dickinson and Company</td><td></td></tr><tr><td>Brandzig Ultra-Fine Insulin Syringes 29G 1cc 1/2&quot; 100-Pack</td><td>CMD 2613</td><td>Brandzig</td><td></td></tr><tr><td>Brilliant Violet 421 anti-mouse CD19 Antibody</td><td>115537</td><td>BioLegend</td><td>50 &amp;micro;g/mL</td></tr><tr><td>CAPS for Flow Tubes w/strainer mesh 35 &amp;micro;m, Dual position for 12 x 75 mm tubes, sterile</td><td>T9009</td><td>Southern Labware</td><td></td></tr><tr><td>Carbon Dioxide USP E CGA 940&amp;nbsp;</td><td>CD USPE</td><td>AirGas USA</td><td></td></tr><tr><td>Cole-Parmer Essentials Low-Form Beaker, Glass, 500 mL</td><td>UX-34502-46</td><td>Cole-Parmer</td><td></td></tr><tr><td>Collagenase 2</td><td>LS004176</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Connector brass chrome plated 1/4&quot; female NPT x 1/4&quot; barb</td><td>Y992611-AG</td><td>AirGas USA</td><td></td></tr><tr><td>Cytek Aurora Flow Cytometer</td><td></td><td>Cytek Biosciences</td><td></td></tr><tr><td>Diss 1080 Nipple 1/4 BARB CP</td><td>M-08-12</td><td>AirGas USA</td><td></td></tr><tr><td>DNase I - 40,000 U</td><td>D4527</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Easypet&amp;nbsp;3 - Electronic Pipette Controller</td><td>4430000018</td><td>Eppendorf</td><td></td></tr><tr><td>Electronic Balance, AX223/E</td><td>30100606</td><td>Ohaus Corp.</td><td></td></tr><tr><td>Eppendorf 5810R centrifuge</td><td>5810R</td><td>Eppendorf</td><td></td></tr><tr><td>Eppendorf Research plus 1-channel variable pipettes</td><td></td><td>Eppendorf</td><td></td></tr><tr><td>FlowJo 10.8.1</td><td></td><td>BD Becton, Dickinson and Company</td><td></td></tr><tr><td>GLACIERbrand, triple density Ice Pan (IPAN-3100)</td><td>Z740287</td><td>Heathrow Scientific</td><td></td></tr><tr><td>HBSS (1x) - Ca2+ [+] Mg2+ [+]</td><td>14025076</td><td>gibco</td><td>1x</td></tr><tr><td>Hyaluronidase</td><td>H3506</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Kelly Hemostats, Straight</td><td>13018-14</td><td>Fine Science Tools</td><td></td></tr><tr><td>Luer Slip Syringe sterile, single use, 20 mL</td><td>302831</td><td>BD Becton, Dickinson and Company</td><td></td></tr><tr><td>M1 Adj. Reg 0-100 PSI/CGA940</td><td>M1-940-PG</td><td>AirGas USA</td><td></td></tr><tr><td>McKesson Underpads, Moderate</td><td>4033-CS150</td><td>McKesson</td><td></td></tr><tr><td>Navigator Multi-Purpose Portable Balance</td><td>NV2201</td><td>Ohaus Corp.</td><td></td></tr><tr><td>PBS pH 7.4 (1X) Ca2+ [-] Mg2+ [-]</td><td>10010023</td><td>gibco</td><td>1x</td></tr><tr><td>PE anti-mouse/human CD45R/B220 Antibody</td><td>103208</td><td>BioLegend</td><td>200 &amp;micro;g/mL</td></tr><tr><td>PerCP/Cyanine5.5 anti-mouse CD45 Antibody</td><td>103132</td><td>BioLegend</td><td>100 &amp;micro;g 500 uL</td></tr><tr><td>Petri dish, Stackable 35 mm x 10 mm Sterile Polystyrene</td><td>FB0875711YZ</td><td>Fisher Scientific</td><td></td></tr><tr><td>Pkgd: Diss 1080 Nut/CO2/CO2-02</td><td>M08-1</td><td>AirGas USA</td><td></td></tr><tr><td>Powerful 6 Watt LED Dual Goose-Neck Illuminator</td><td>LED-6W</td><td>AmScope</td><td></td></tr><tr><td>PrecisionGlide Needle 25 G x 5/8 (0.5 mm x 16 mm)</td><td>305122</td><td>BD Becton, Dickinson and Company</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) Clone 2.4G2 (RUO)</td><td>553141</td><td>BD Becton, Dickinson and Company Biosciences</td><td>0.5 mg/mL</td></tr><tr><td>R 4.1.1</td><td></td><td>The R Foundation</td><td></td></tr><tr><td>Razor Blades</td><td>9501250000</td><td>Accutec Blades Inc</td><td></td></tr><tr><td>Regulator analytical two stage 0-25 psi delivery CGA320 3500 psi inlet</td><td>Y12244A320-AG</td><td>AirGas USA</td><td></td></tr><tr><td>Rotor A-4-62, incl. 4 x 250 mL rectangular buckets</td><td>Rotor A-4-62</td><td>Eppendorf</td><td></td></tr><tr><td>Serological pipette, plugged, 10 mL, sterile, non-pyrogenic/endotoxin-free, non-cytotoxic, 1 piece(s)/blister</td><td>86.1254.001</td><td>Sarstedt AG &amp;amp; Co KG</td><td></td></tr><tr><td>Sigma label tape</td><td>L8394</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>SpectroFlo 3.0.0</td><td></td><td>Cytek Biosciences</td><td></td></tr><tr><td>Spex VapLock Luer Fitting, PP, Straight, Male Luer Lock x 1/8&quot; Hose Barb; 1/EA</td><td>MTLL230-6005</td><td>Spex</td><td></td></tr><tr><td>Std Wall Lab Tubing, Size S2, Excelon, 1/8&quot; ID x 3/16&quot; OD x 1/32&quot; Wall x 50&#039; Long</td><td>CG-730-003</td><td>Excelon Laboratory</td><td></td></tr><tr><td>Syringe PP/PE without needle, 3 mL</td><td>Z683566</td><td>Millipore Sigma</td><td></td></tr><tr><td>Syringe pump</td><td>55-1199 (95-240)</td><td>Harvard Apparatus</td><td></td></tr><tr><td>Thomas 3-Channel Alarm Timer TM10500</td><td>9371W13</td><td>Thomas Scientific</td><td></td></tr><tr><td>Tube Rack, 12&amp;nbsp;positions, 6 for 5.0&amp;nbsp;mL and 15&amp;nbsp;mL tubes and 6 for 25&amp;nbsp;mL and 50&amp;nbsp;mL tubes, polypropylene, numbered positions, autoclavable</td><td>30119835</td><td>Eppendorf</td><td></td></tr><tr><td>Tube Rack, 12&amp;nbsp;positions, for 5.0&amp;nbsp;mL and 15&amp;nbsp;mL tubes, polypropylene, numbered positions, autoclavable</td><td>30119827</td><td>Eppendorf</td><td></td></tr><tr><td>TYGON R-3603 Laboratory Tubing, I.D. &amp;times; O.D. 1/4 in. &amp;times; 3/8 in.</td><td>T8913 (Millipore Sigma)</td><td>Tygon, Saint-Gobain</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>SI-0236</td><td>Scientific Industries, Inc.</td><td></td></tr><tr><td>VWR Dissecting Forceps with Guide Pin with Curved Tips</td><td>89259-946</td><td>Avantor, by VWR</td><td></td></tr><tr><td>VWR Dissecting Scissors, Sharp Tip, 4&amp;frac12;&quot;</td><td>82027-578</td><td>Avantor, by VWR</td><td></td></tr><tr><td>VWR Incubating Orbital Shaker, Model 3500I</td><td>12620-946</td><td>Avantor, by VWR</td><td></td></tr><tr><td>Zombie Aqua Fixable Viability Kit</td><td>423102</td><td>BioLegend</td><td></td></tr></tbody></table>

flow cytometry-based quantification and analysis of myocardial b-cells

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

Once the acquisition is complete and all the events are collected, the data should be analyzed according to standard flow cytometry practice. The focus of the analysis will vary depending on the individual goal of each experiment. In this case, quantification of intravascular and extravascular B-cells was pursued, and it was expressed as the number of cells per mg of tissue.
When using a spectral cytometer, it is recommended to start the analysis with an initial gating to remove debris with si...

Watch this Scientific Journal Video about Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells at JoVE.com

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

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

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

flow-cytometry-based-quantification-and-analysis-of-myocardial-b-cells

Research

JoVE Journal

Immunology and Infection

2.4K Views.  The Johns Hopkins University School of Medicine. Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

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

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Developmental Biology

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 ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

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 ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Biology

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...