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>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 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>
		</tr>
		<tr>
			<td>Aluminum Foil 12 in x 75&#39; 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&#34; 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 &#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>
		</tr>
		<tr>
			<td>Carbon Dioxide USP E CGA 940&#160;</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&#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>
		</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 &#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>
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		<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>
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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

2.3K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...