Name: flow cytometry-based quantification and analysis of myocardial b-cells
Uploaded: 2022-08-17T14:50:00
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 a...

<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. 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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" 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 700 anti-mouse/human CD11b Antibody</td>
			<td>101222</td>
			<td>BioLegend</td>
			<td>100 &#181;g 200 &#181;L</td>
		</tr>
		<tr>
			<td>(CellTreat 29481) Cell Strainer, 40 &#181;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 &#181;L Graduated TipOne Filter Tips</td>
			<td>11213810</td>
			<td>USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;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 &#181;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 &#8211; 7 mL, for 250 mL rectangular bucket in Rotor A-4-62</td>
			<td>22638246</td>
			<td>Eppendorf</td>
			<td></td>
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			<td>UPC 109153</td>
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			<td>Anesthesia Induction Chamber - Mouse</td>
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			<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&#34; 100-Pack</td>
			<td>CMD 2613</td>
			<td>Brandzig</td>
			<td></td>
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			<td>Brilliant Violet 421 anti-mouse CD19 Antibody</td>
			<td>115537</td>
			<td>BioLegend</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>CAPS for Flow Tubes w/strainer mesh 35 &#181;m, Dual position for 12 x 75 mm tubes, sterile</td>
			<td>T9009</td>
			<td>Southern Labware</td>
			<td></td>
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		<tr>
			<td>Carbon Dioxide USP E CGA 940&#160;</td>
			<td>CD USPE</td>
			<td>AirGas USA</td>
			<td></td>
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		<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>
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		<tr>
			<td>Connector brass chrome plated 1/4&#34; female NPT x 1/4&#34; 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&#160;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 &#181;g/mL</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-mouse CD45 Antibody</td>
			<td>103132</td>
			<td>BioLegend</td>
			<td>100 &#181;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>
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		<tr>
			<td>Razor Blades</td>
			<td>9501250000</td>
			<td>Accutec Blades Inc</td>
			<td></td>
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		<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 &#38; 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&#34; 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&#34; ID x 3/16&#34; OD x 1/32&#34; Wall x 50&#39; 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&#160;positions, 6 for 5.0&#160;mL and 15&#160;mL tubes and 6 for 25&#160;mL and 50&#160;mL tubes, polypropylene, numbered positions, autoclavable</td>
			<td>30119835</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rack, 12&#160;positions, for 5.0&#160;mL and 15&#160;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. &#215; O.D. 1/4 in. &#215; 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&#189;&#34;</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. ...

The literature reports contrasting data on the prevalence of myocardial B-cells. This likely stems from variability in perfusion and digestion techniques. Our protocol empowers reproducible flow cytometry-based analysis on myocardial B-cells.This technique maximizes the recovery yield of B-cells and reduces variability in flow cytometry counts among B-cells and other populations from heart samples. This optimized digestion and isolation method can be easily modified to implement an extended antibody panel to study different immune cell types in the heart. The protocol can also be applied to other organs, such as lung or liver, to study B-cells associated with those organs.To begin, weigh a 12-week-old male mouse. Load a 310-cc insulin syringe with 100 microliters of the B220 antibody solution. Inject the diluted B220 antibody by retro-orbital injection and promptly proceed to organ harvesting and perfusion.After euthanasia, hold the skin of the mouse with forceps at the epigastric area. Make a two-millimeter opening in the skin using scissors and use the forceps to peel the skin away to reveal the fascia layer. Cut at the epigastrium through the fascia and peritoneum.Clamp the xiphoid process using hemostatic forceps. Make a vertical cut through the chest wall at the level of the midclavicular line on each side and lift the anterior chest wall to reveal the mediastinum content. Hold the aorta securely from behind using forceps.Introduce the syringe needle into the apex of the heart. Perfused with three milliliters of HBSS at a rate of three milliliters per minute. Cut the aorta and remove the heart.Wash the heart in the Petri dish with HBSS. Weigh the isolated heart and note the weight in milligrams. Mince the heart at room temperature into small pieces with a blade.The heart pieces should be about 1.5 millimeters in size. Measure the mass of heart tissue using a balance and place 60 milligrams of the minced heart to be digested in a 15-milliliter tube on ice with three milliliters of HBSS. Repeat this process for a mouse that did not receive the B220 antibody injection and place it in the tube with three milliliters of HBSS labeled reference control tissue.Add enzymes to each tube containing minced tissues. Add 0.5 microliters per milligrams of DNase I, one 0.5 microliters per milligram of collagenase II, and 0.2 microliters per milligram of hyaluronidase. Place the digestion reaction in the shaker for 30 minutes at 300 rotations per minute.Tubes are placed in the shaker at a small angle, about 25 degrees of incline. Add seven milliliters of HBSS to each of the 15-milliliter reaction tubes and centrifuge at 250 G for five minutes at four degrees Celsius. After decanting the supernatant, resuspend each pellet in five milliliters of ACK lysis buffer.Incubate for five minutes at room temperature. After adding 10 milliliters of PBS to each tube, mix and filter into a 50-milliliter tube with a 40-micrometer strainer. Smash any debris on the filter with a syringe plunger to ensure that all the material passes through the filter.After adding PBS through the filter until the volume reaches 25 milliliters per heart, add an additional 25 milliliters of PBS to the reference control tissue tube and divide the volume into two different tubes. Label one of the tubes unstained and the other live/dead. After centrifuging at 250 G for five minutes at four degrees Celsius, decant the supernatant and resuspend the unstained tube in 100 microliters of PBS and each of the other pellets in 100 microliters of the live/dead stain solution.Incubate on ice for 30 minutes in the dark. After adding 500 microliters of FACS buffer to each sample, transfer it into a FACS tube through a 40-micrometer filter. After refusing the FACS tubes at 250 G for five minutes at four degrees Celsius and discarding the supernatant, resuspend the pellet in 500 microliters of FACS buffer.Add 50 microliters of FC blocking solution to each tube, except for the unstained and live/dead tubes. Mix and incubate for five minutes. After adding 50 microliters of antibody mix solution to each tube, except for the unstained and live/dead tubes, incubate for 30 minutes in the dark, covering the ice bucket with aluminum foil.As with the live/dead stain, wash again with 500 microliters of FACS buffer and resuspend in 300 to 500 microliters of FACS buffer. Open the instrument control software. At the top of the window, select the Acquisition tab and click on New.In the library section, in the field Type to filter, type PE, PerCP-Cy5.5, brilliant violet 421, Alexa Fluor 700, and Zombie Aqua, selecting the fluoro-4 and clicking Add after typing each. Then, click Next to proceed to the Groups tab. Now click on the symbol with a tube to add the heart for the analysis.Click on the reference group and a window will display. In the fluorescent tags column, select Zombie Aqua as the fluorescent tag, deleting any others by clicking on the trash red icon next to each. Then, in the Control Type icon, select Cells.Then, click Save. Click Next. Then, click the Markers tab.A table will display. Type the corresponding cell marker in the first row of each fluoro-4 column, and press Enter. Click Next twice to go to the Acquisition tab.In Events To Record of the experimental samples, type 10 million, and in the same field for the reference group line, type 200, 000. Click Save and Open. On the bottom left, click on Instrument Control.Set voltage to FSC 50, SSC 175, SSC-B 175. Select the Acquisition Control. For the flow rate option, select High from the dropdown menu.Vortex the unstained heart tube and place it securely on the sample injection port. Ensure the green indicator arrow is pointing to the correct sample. Then, click Record and wait until the events are recorded.Do the same for the live/dead stained heart tissue. Select the Unmix menu and set the controls. Select the checkbox for Autofluorescence as a Fluorescent Tag.Then, click Next to proceed to the Identify Positive/Negative Populations tab. In this tab, a plot of forward scatter area versus side scatter area will be displayed. Click and drag the points of the polygon to select an area between 1 and 4 million on the forward scatter area axis and between 0.2 and 3 million on the side scatter area axis.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 the unstained, no positive/negative must be selected. Click the Live Unmixing button. Now acquire the experimental samples, vortex the tube, and place it securely on the SIP.Ensure the green indicator arrow is pointing to the correct sample. Then, click Record in the Acquisition Control panel of the cytometer software and wait until the events are recorded. Select the Default Unmixed Worksheet tab.Create an FCS-A versus SSC-A plot using the dot plot tool on top. Select events higher than 0.4 million on the FSC-A axis and higher than 0.8 million on the SSC-A axis using the polygon selection tool. Create a new plot by double-clicking the selection, and in this new plot, right click on the Y-axis label and select live/dead stain.Right click on the X-axis label and select Autofluorescence A.Select all the events below 10 to the fifth on the live/dead axis. These are the living cells. Double-click in this selection.In the new plot, select PerCP-Cy5.5A for the X-axis and SSC-A for the Y-axis. Select the populations on the right side of the PerCP-Cy5.5A. These are the immune cells.Double-click on the selection. On the new plot, select FSC-A for the X-axis and forward scatter height for the Y-axis. Select the events that follow a trend in which the FSC-A value and SSC-A value are the same.This selects single cells and eliminates doublets. Double-click on this selection to display a new plot in the CD45-positive single-cell population. From the CD45-positive single-cell population plot, select brilliant violet 421 on the X-axis versus SSC-A on the Y-axis.Select the population to the right on the brilliant violet 421 axis. This is the B-cell population. Double-click twice in this population to create two new plots with the B-cell population.Set up the first B-cell plot with PEA on the X-axis and brilliant violet 421A on the Y-axis. The population to the right corresponds to the intervascular B-cells, and the population to the left represents the interstitial B-cells. Select both.Set up the second B-cell plot with Alexa Fluor 700A on the X-axis and SSC-A on the Y-axis. The population on the left corresponds to the CD11b-negative cells, and the events to the right correspond to the CD11b-positive cells. Right click on each section and then click Gate properties.Click on Count and Parent to display the numbers of events and percentages of the parent populations. Record the number of events and percentages for further data analysis. After the removal of doublets in a naive uninjured heart, it's expected that about 20%of CD45-positive cells are going to be CD19-positive B-cells.Of these cells, on average, 5.5%are located in the interstitial space. 94.5%are in the intravascular space. From the whole B-cell population found in the heart, only about 1.6%corresponds to B1 cells based on the surface marker CD11b and the other 98.4%corresponds to B2 cells.When analyzing myocardial B-cells via flow cytometry, it is crucial to perfuse and mince the heart properly following the protocol. Proper filtration of the digested heart is also essential to maximize the recovery of immune cells.

Research

JoVE Journal

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