Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

1. Preparation

1. Before beginning, put on laboratory gloves and the appropriate protective clothing.
2. Sterilize all the dissection tools, first with a detergent and then with 70% ethanol and then dry thoroughly.
3. Prepare 50mL of Hank's balanced salt solution (HBSS) containing 2% fetal calf serum (FCS).

2. Dissection

1. Using a carbon dioxide delivery system, euthanize the mouse by hypoxia. Secure the euthanized mouse on a dissection plate in the supine position and perform a longitudinal laparotomy using scissors and forceps.
2. Using forceps, move the intestines and stomach on the right side of the abdomen to expose the stomach and spleen. The spleen is attached to the stomach.
3. Using forceps, carefully detach the spleen from the stomach and place it in the Petri dish containing 5mL of HBSS 2% FCS.

3. Immune Cell Isolation

1. Place the spleen in a 40µm cell strainer over the same Petri dish. Crush the spleen with a plunger to dissociate it in the same dish.
2. Transfer the dissociated spleen and the fluid into a 15mL centrifuge tube.
3. Centrifuge the tube at 370 x g for 7 min at 10°C and discard the supernatant, avoiding the pellet.
4. Resuspend the pellet in 2mL of potassium acetate to lyse the erythrocytes. Wait for 2 min and then make up the volume up to 15mL using HBSS 2% FCS.
5. Centrifuge the tube again at 370 x g for 7 min at 10°C. Discard the supernatant and resuspend the pellet in 5mL of HBSS 2% FCS.
6. Count the cells using a trypan blue staining assay and adjust the final cell concentration to 10⁷ cells/mL using appropriate volume of HBSS 2% FCS.

4. Cell Staining

1. Transfer 200µL of the cell suspension into six FACS tubes, labelled 1 to 6.
2. Centrifuge the tubes at 370 x g for 7 min at 10°C and discard the supernatant avoiding the pellet.
3. Next, prepare six novel antibody mixes by adding appropriate amount of antibody to 200µL HBSS 2% FCS according to Table 1.
4. Then, transfer these antibody mixes to the corresponding numbered FACS tubes.
5. Incubate the cell suspensions mixed with the antibodies for 20 minutes on ice in the dark.
6. Add 1mL of HBSS 2% FCS to each tube and then centrifuge again at 370 x g for 3 min at 10°C.
7. Discard the supernatant and resuspend the pellet in 200µL of HBSS 2% FCS.
8. Transfer the resuspended pellets to new FACS tubes.

Table 1: Antibodies mix composition. Six mixes of 200µL of HBSS + antibody were prepared for the experiment. Mix 1 is for PMT setting, mixes 2 through 5 are for compensation settings, and mix 6 is for cell sorting.

5. FACS Calibration

1. First, turn on the sorter and perform "Fluidics Startup."
2. Turn on the stream and then wait for 15 min for the stream to stabilize.
3. Adjust the amplitude of the stream to obtain detached drop formation. Then click the "Sweet Spot" to complete the amplitude adjustment.
4. Put the neutral density (N.D.) filter - 1.0 and open the "CST" interface which stands for cytometer setup and tracking.
5. To perform daily quality control, first dilute the CST beads with FACS medium following the manufacturer's instructions and then, perform the CST control.
6. Once CST control is complete, replace the N.D. 1.0 filter with the N.D. 2.0 filter on the cytometer.
7. Next, dilute drop delay beads in FACS medium following the manufacturer's instructions and then load into the FACS.
8. To ensure proper sorting perform Drop Delay-
   1. First click "Voltage" and then "Optical Filter". The right quadrant should be equal to 100%. If necessary, adjust the red laser screw on the cytometer left or right to obtain 100% in the right quadrant.
   2. Next, perform a test sort to ensure that the stream falls into the collection tube. To do so, click on "Waste Drawer" and start "Test Sort". Check that side streams fall into collection tubes. If they do not, adjust voltage until they do.
   3. Navigate to the experimental template. Then, open the "Accudrop Drop Delay" experiment and click the "Sorting Layout".
   4. Change the flow rate to obtain 1000 to 3000 events per second.
   5. Click "Voltage" and then "Optical Filter". The left quadrant should be equal to 0 and right quadrant to 100.
   6. In the "Sort Layout" window click on "Sort" and then click "Cancel". The left quadrant should be equal to 100 and right quadrant to 0. If left quadrant is less than 95 click "Auto Delay" to automatically adjust it.

6. Flow Cytometry and Purity Control

1. Begin flow cytometry by starting with the tube 1 (unstained cells) to define the cell morphology and the negative peaks of the fluorochromes. Set up the forward and side scatter and define the voltages of each fluorescent parameter. Place the negative population in the first decade by using the grids on each dot plot.
2. Next, load tube 2 (single color control) in the cytometer. Adjust the spectral overlap until the negative and positive population medians are aligned or use the automatic calculation software. It is important to keep the signal on scale. Compensation controls must match the experimental fluorochromes and detectors settings. Record 10,000 events.
3. Repeat these steps with tube 3, 4, and 5 (other single color controls).
4. Next, load tube 6 (multi-stained cells) and define cell populations of interest by using a specific gating strategy.
5. In Sorting Layout window, select the cell population of interest. Select the cell threshold in the "Target Events" and the precision level in the "Precision". Here only one population is being sorted, however, four different populations can be sorted at the same time.
6. Once ready click "Sort" and "OK", then wait for cell sorting.
7. Once cell sorting is complete, perform a purity control by first pipetting 10µL of sorted cells into a new FACS tube with 90 microliters of HBSS 2% FCS.
8. Then, load the tube the cytometer. Record and analyze the phenotypes of the cells to verify that the gating strategy worked as intended.

7. Data Analysis

1. Open the 'FlowJo' software and drag the files for each tube in "All sample" window.
2. Double click on a file to open it in a new window.
3. Click on the "Polygon" and recreate gating strategy used previously.
4. Repeat the steps with all the other files.
5. To visualize the dot plots, click on "Layout editor" and drag the populations of interest from tube 6 and purity control in the layout editor tab. Cells should only appear in the population of interest in purity control (see Figure 2).
6. To check the purity of B lymphocytes in the sorted cells, click on "Table editor". Drag B lymphocyte population from tube 6 and purity control in the table.
7. On "Statistic" menu select the frequency of CD45⁺ cells to test the purity of this cell population, then click on "Create table".
8. Parameter values appear in a new table. In purity control window, check the frequency of of B lymphocytes within the CD45⁺ cells, which should be higher than 98% (see figure 2, bottom panel).