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

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.