Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

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 wipe them dry thoroughly.
3. Prepare 50 mL 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 5 mL of HBSS 2% FCS.

3. Immune Cell Isolation

1. Place the spleen on a 40 µm cell strainer over the same Petri dish. Crush the spleen with a plunger to dissociate it.
2. Transfer the dissociated spleen and the fluid into a 15 mL 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 2 mL of potassium acetate to lyse the erythrocytes. Wait for 2 min and then make up the volume up to 15 mL 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 5 mL 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 an appropriate volume of HBSS 2% FCS.

4. CFSE Staining and T-cell Stimulation

1. Distribute 10⁷ isolated spleen cells/tube in 4 tubes (15 mL tubes, labeled 1 to 4)
2. Add 3 mL HBSS 2%FCS to each tube.
3. Add 1 µL of CSFE in each tube (final concentration- 5 µM).
4. Incubate the tubes at 37°C in a 5% CO₂ incubator for 10 min.
5. In tubes 3 and 4, add 12 mL of HBSS 2% FCS + anti-CD3 antibody at a final concentration of 2.5 µg/mL. Tubes 3 and 4 are be stimulated using anti-CD3 antibody, to observe the effect on the cell cycle.
6. In tubes 1 and 2 add 12 mL of HBSS 2% FCS. The cells in tubes 1 and 2 will not be stimulated.
7. Centrifuge all the tubes at 370 x g for 7 min at 10°C. Discard the supernatants.
8. Resuspend the pellets in 2 mL of HBSS 2%FCS.
9. Transfer the resulting solutions into separate wells on a 6-well plate.
10. Incubate the cells at 37°C, 5% CO₂ for 3 days.

5. Cell Staining

1. On day 3, add 2 mL HBSS 2%FCS in well 1 and 3.
2. Pipet vigorously and transfer the samples into 5 mL FACS tubes.
3. Continue incubating the remaining cells from wells 2-4 at 37°C, 5% CO₂. They will be analyzed on day 5 to investigate the long-term effects of stimulation on the cell cycle.
4. Centrifuge the tubes at 370 x g for 7 min at 10°C. Discard the supernatants.
5. Add 100 µL of antibody mix (see Table 1) to each tube.

Antibody	Fluorochrome	Dilution
CD3	Pacific Blue	1/100
CD4	BV786	1/1600
CD8	PE	1/400
Thy1.2	BV605	1/400

Table 1: Antibody mix composition. Four antibody cocktail preparations using concentrated antibody-fluorescent conjugates and HBSS.

6. Incubate the tubes for 20 min on ice in the dark.
7. Add 1 mL of HBSS 2%FCS and centrifuge the tubes at 370 x g for 7 min at 10°C.
8. Discard the supernatant and resuspend the pellets in 200 µL of HBSS 2% FCS.
9. Transfer the resuspended pellets to new, labelled FACS tubes.
10. Evaluate T-cell proliferation using FACS.
11. On day 5, repeat the cell staining process with the cells from the remaining two wells of the 6-well plate.

6. Data Analysis

1. Open the "FlowJo" software and drag the files into the "All sample" window.
2. Double click on the file for unstimulated cells collected on day 3 to display a dot plot with forward scatter on the Y axis and side scatter on the X axis.
3. Click on "Polygon" and create a gating strategy to select lymphoid cells, Thy1.2⁺ CD3⁺ cells and distinguish CD4⁺ and CD8⁺ cells (see Figure 1).

Figure 1: Gating strategy. Cells are first gated based on their morphology (left: FSC-A, SSC-A). T cells are then gated (middle: CD3, Thy1.2) and further divided on CD4⁺ T cells (orange) and CD8⁺ T cells (blue) (right: CD4, CD8). Please click here to view a larger version of this figure.

4. Repeat the steps with other files.
5. To determine the frequencies of dividing and non-dividing cells, first visualize the cell populations by clicking on "Layout editor."
6. Drag the CD4 T cells and CD8 T cells from each of the four tubes to the "All sample window"
7. Graphs representing each population will appear.
8. Use histogram to visualize the results and select "CFSE" as parameter for comparison of the different tubes and the different populations.
9. Non-dividing cells maintain higher levels of CFSE, whereas proliferating cells split the content of CFSE to dividing cells
10. Create a gate on non-dividing cells and apply it in all tubes.
11. Create a gate on dividing cells and apply it in all tubes.
12. To examine the frequency of dividing CD3+ cells, click on "Table editor."
13. Drag the populations of interest - dividing CD8 T-cells and dividing CD4 T-cells - into "Table."
14. On the "Statistic" menu, select "frequency of T cells," then click on "Create table" to reveal the frequency in a new table.
15. Click on "Create table." To reveal the frequency values, appear on a new table.