An Assay for Cell Cycle Analysis of Antigen-Specific CD8⁺ T Cells

1. Preparation of medium and staining solution

1. Prepare Complete Medium: Roswell Park Memorial Institute (RPMI) medium with 2 mM glutamine, 100 U/mL penicillin/streptomycin, 50 µM beta-mercaptoethanol, and 10% volume/volume (v/v) of fetal bovine serum (FBS)
2. Prepare Staining Buffer: Phosphate-buffered saline without Ca²⁺/Mg²⁺ (PBS) with 1% weight/volume (w/v) bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid disodium salt (EDTA)

2. Mouse treatment

1. Prime 7-8-week-old, female Balb/c mice by intramuscular (i.m.) injection in the quadriceps of the human immunodeficiency virus (HIV)-1-gag-expressing-chimpanzee adenoviral vector (ChAd3-gag) with a dose of 10⁷ viral particles.
2. At 1-4 months after priming, boost once the mice by i.m. injection of HIV-1-gag-expressing modified vaccinia Ankara virus (MVA-gag) with a dose of 10⁶ plaque-forming units.
3. At day 3 post-boost, sacrifice the boosted mice by cervical dislocation, and analyze them in parallel with untreated mice.
4. Harvest the lymph nodes (LNs) draining the quadriceps (iliac, popliteal, and inguinal) and the spleens from boosted and untreated mice. Furthermore, collect the bone marrow (BM) from the two hind legs from untreated mice, and use this BM for flow cytometer settings and as positive control for cell cycle analysis (Figure 1).
   NOTE: Generate ChAd3-gag and MVA-gag vectors as described previously.

3. Isolation of draining LN, spleen

1. Isolation of spleen and LN cells
   1. Place 5 mL of complete medium in each of two 15 mL tubes, and keep them on ice, ready for organs to be collected.
   2. Sacrifice an adult mouse by cervical dislocation.
   3. Place the mouse on its back, and sterilize the skin surface with 70% v/v ethanol.
   4. To collect inguinal LNs, make a ~1 cm longitudinal incision on the abdomen with scissors, and stretch the incision with the forceps.
   5. Visualize inguinal LNs on the internal surface of the skin, and harvest them with the forceps. Place the inguinal LNs in one of the two 15 mL tubes prepared in step 3.1.1.
   6. To collect the spleen, make a peritoneal incision with scissors and remove the spleen. After cutting the surrounding connective tissue, place the spleen into the second 15 mL tube prepared in step 3.1.1.
   7. To collect iliac LNs, move the bowels aside and visualize iliac LNs close to the inferior vena cava, and then collect them by using the forceps. Place the iliac LNs in the same tube containing the inguinal LNs.
      ​NOTE: To obtain enough LN cells for staining (see section 4), it is often necessary to pool popliteal, inguinal, and iliac LNs from one mouse. These LNs are all draining the quadriceps (the site of i.m. vaccination). This protocol uses only one 15 mL tube of pooled LNs.
   8. To collect popliteal LNs, grasp the skin of the hind legs and gently pull it downwards to uncover the muscles. Then, insert the forceps between the muscles under the knee joint, and collect the popliteal LNs. Place the popliteal LNs in the same tube containing inguinal and iliac LNs.
      ​NOTE: See note after 3.1.7.
   9. Place the spleen into a 70 µm cell strainer within a 60 mm culture dish filled with 5 mL of complete medium. Using a 5 mL syringe plunger, gently mash the organ until its complete disaggregation.
   10. Remove the strainer, and transfer the cell suspension to a clean 15 mL tube.
   11. Add 5 mL of complete medium to the culture dish, and carefully wash the dish and the strainer to ensure that all cells have been recovered. Pool with the rest of the spleen cell suspension into the 15 mL tube.
   12. For the pooled inguinal, iliac, and popliteal LNs, prepare a single-cell suspension following a similar procedure to that used in steps 3.1.9 to 3.1.11 for the spleen.
   13. Centrifuge cells at 400 × g for 10 min at 4 °C. Discard the supernatant and resuspend the cell pellets in PBS.
   14. Count the cells with a Neubauer chamber using red blood cell lysis buffer and 0.04% v/v trypan blue in PBS.

4. Staining of spleen, LN​

1. Divide cell samples to be stained into 3 subgroups: cell samples for compensation, including BM cells from untreated mice to be stained only with Hoechst 33342 (henceforth referred to as Hoechst), and spleen cells from untreated mice to be used to prepare a dead/live cell mix for dead cell dye compensation; positive control for cell cycle analysis, consisting of a BM sample from untreated mice; and experimental samples containing spleen and LN samples from untreated and vaccinated mice.
   NOTE: Ensure that there are enough spleen and LN cells for analysis of sufficient numbers of gag-specific CD8 T cells. It is often necessary to use pooled spleen cells and pooled LN cells from 3 vaccinated mice and stain two or more identical samples of pooled cells, each containing 3 × 10⁶ cells. Merge identical samples at the step of Hoechst staining. Similarly, stain pooled spleen cells and LN cells from 3 untreated mice, and merge identical samples at the end. Set aside an unstained sample of spleen cells from an untreated mouse to be used for instrument and compensation setup.
2. Prepare dead/live cell mix for dead cell dye compensation (this mix of cells will be stained only with the dead cell dye).
   1. Heat a water bath at 65 °C.
   2. Take an aliquot of spleen cells (~3 × 10⁶).
   3. Transfer the cell suspension to a microfuge tube, place it in the water bath at 65 °C for 5 min, and then immediately place it on ice for 10 min.
   4. Mix the heat-killed cells with live spleen cells (~3 × 10⁶) in a ratio of 1:1, and transfer half of the mixture to a 96 well-round bottom plate (~3 × 10⁶ cells/well for the dead cell staining control).
3. Dead cell staining of experimental samples, positive control for cell cycle analysis, and dead/live cell mix
   1. Transfer spleen, LN, BM cells (3 × 10⁶ cells/well), and the dead/live cell mix (section 4.2) into a 96-well round-bottom plate, according to the staining scheme (step 4.1), and centrifuge at 400 × g for 3 min at 4 °C.
   2. Resuspend each cell pellet in 50 µL of dead cell dye diluted in PBS, and resuspend by pipetting up and down 3 times immediately.
   3. Incubate for 30 min at 4 °C, protected from light.
   4. Wash cells 2 times with staining buffer; the first time with 200 µL and the second time with 250 µL. For each wash centrifuge the plate at 400 × g for 3 min at 4 °C.
   5. Discard the supernatant, and resuspend the cell pellet in 20 µL of PBS.
4. Membrane cell staining with major histocompatibility complex (MHC)-peptide multimers and monoclonal antibodies (mAbs).
   1. Taking into account the necessary volumes according to the staining scheme (Flow cytometer settings, Table 1), prepare the following reagents:
      1. Dilute mAb 2.4G2 in the staining buffer according to the appropriate dilution (see Table of Materials); for each sample to be stained, use 10 µL of this dilution.
         NOTE: 2.4G2 mAb blocks non-antigen-specific binding of immunoglobulins to the FcγIII and FcγII receptors.
      2. Dilute the H-2k(d) AMQMLKETI allophycocyanin (APC)-labeled tetramer (Tetr-gag) in the staining buffer to obtain the appropriate dilution (see Table of Materials); for each sample to be stained, use 20 µL of this dilution.
      3. Prepare the antibody mix by diluting mAbs in the staining buffer according to the appropriate dilution (see Table of Materials) that has been previously determined in titration experiments; for each sample to be stained, use 20 µL of this antibody mix.
         NOTE: Here, anti-CD3e peridinin chlorophyll protein (PerCP-Cy5.5) (clone 145-2C11), anti-CD8a brilliant ultraviolet (BUV805) (clone 53-6.7), and anti-CD62L phycoerythrin cyanine7 (PECy7) (clone MEL-14) were used.
   2. Add 10 µL of the previously diluted 2.4G2 mAb (step 4.4.1.1), and incubate for 10 min at 4 °C, protected from light.
   3. Add 20 µL of the previously diluted Tetr-gag APC (step 4.4.1.2) and 10 µL of H-2k(d) AMQMLKETI phycoerythrin (PE) pentamer (pent-gag). Incubate for 15 min at 4 °C, protected from light.
   4. Add 20 µL of the previously prepared antibody mix (step 4.4.1.3), and incubate for 15 min at 4 °C, protected from light.
      NOTE: Hence, the final volume is 80 µL per well (step 4.3.5, steps 4.4.2 to 4.4.4).
   5. Wash cells with 200 µL of staining buffer. Centrifuge at 400 × g for 5 min at 4 °C.
   6. Resuspend the cell pellet in 250 µL of staining buffer, and transfer the cell suspension to 5 mL tubes. Add 1 mL of staining buffer to the tube, and centrifuge at 400 × g for 5 min at 4 °C.
   7. Take the aliquot of BM cells (3 × 10⁶ cells) (see list of cell samples, section 4.1) to be used to compensate the Hoechst channel (Hoechst 33342 is excited by an ultraviolet laser (flow cytometer settings (Table 2)), and transfer the cell suspension into a 5 mL tube. Add 1 mL of staining buffer to the tube, and centrifuge 400 × g for 5 min at 4 °C.

5. Fixation/permeabilization

1. Prepare fresh fixation/permeabilization buffer by diluting 1 part of fixation/permeabilization concentrate with 3 parts of fixation/permeabilization diluent, according to the manufacturer's instructions.
2. Discard the supernatant and pulse vortex the samples to completely disaggregate the pellet.
3. Add 1 mL of the freshly prepared fixation/permeabilization buffer to each tube, including a tube with unstained spleen cells (3 x 10⁶, see list of cell samples, section 4.1), and vortex.
4. Incubate for 16 h at 4 °C.
   NOTE: The protocol can be paused here.

6. Intracellular staining

1. Ki67 staining
   1. Prepare fresh permeabilization buffer 1x by diluting permeabilization buffer 10x with distilled water, according to the manufacturer's instructions. Before use, the permeabilization buffer 1x must be filtered through a 0.45 μm filter to eliminate aggregates.
   2. Dilute mAb Ki67 fluorescein isothiocyanate (FITC) (clone SolA15) in permeabilization buffer 1x (see Table of Materials), as determined previously in titration experiments (final volume of 100 μL per sample).
   3. Add 3 mL of permeabilization buffer 1x to each tube, and centrifuge at 400 × g for 5 min at room temperature (RT).
   4. Discard the supernatant and repeat step 6.1.3.
   5. Discard the supernatant, and resuspend the cell pellet in 100 µL of previously diluted mAb Ki67 FITC (step 6.1.2).
   6. Incubate for 30 min at RT, protected from light.
   7. Wash cells 2 times with 4 mL of permeabilization buffer 1x. For each wash centrifuge at 400 × g for 5 min at RT.
   8. Resuspend the cell pellet in PBS considering the following volumes: 350 µL of PBS for the samples to be acquired directly at the flow cytometer; 250 µL of PBS for the samples to be incubated with Hoechst shortly before flow cytometry (section 6.2).
2. DNA staining
   1. Add 250 µL of 4 µg/mL Hoechst in PBS to each sample (the final concentration of Hoechst is 2 µg/mL).
      ​​NOTE: In case two or more identical samples of 250 µL in PBS were prepared, merge them at this step, and add an equal volume of 4 µg/mL Hoechst solution in PBS (final concentration of Hoechst is 2 µg/mL). The number of cells greatly influences the DNA staining step. Use the same cell number in each sample. Be aware that even a slightly reduced cell number (e.g., due to cell loss in previous washing steps) results in higher Hoechst binding to DNA and higher Hoechst intensity.
   2. Incubate for 15 min at RT, protected from light.
   3. Centrifuge the samples at 400 × g for 5 min at RT.
   4. Resuspend the cell pellet in 350 µL of PBS.

7. Preparation of compensation bead samples

1. Prepare 5 µL of the antibody by diluting mAb in the staining buffer appropriately.
   NOTE: For each fluorochrome-conjugated mAb used in the experiment, prepare its corresponding compensation bead sample.
2. Vortex Negative Control and Anti-Rat/Hamster Ig,κ Comp Beads before use.
3. For each sample, introduce one drop (~20 μL) of Negative Control CompBeads and one drop of Anti-Rat/Hamster Ig,k CompBeads.
4. Add 5 µL of the prediluted antibody (step 7.1) to the tube, and pipet up and down.
5. Incubate for 15 min at 4 °C, protected from light.
6. Wash samples with 2 mL of staining buffer. Centrifuge at 400 × g for 5 min at 4 °C.
7. Discard the supernatant, and resuspend the pellet by adding 500 µL of PBS to each tube and vortex.

8. Instrument and compensation setup and experimental sample acquisition at the flow cytometer

NOTE: Refer to flow cytometer settings (Table 2) for the cytometer configuration.

1. General instrument and compensation setup
   1. Open the software for sample acquisition (see Table of Materials), and create a new experiment by clicking New Experiment in the workspace ribbon section and selecting New Blank Experiment.
   2. Double-click on the created experiment to open it.
   3. In the Cytometer Settings window, click on Parameters and select all the channels (e.g., PE, APC, etc.) used in the staining panel including Forward Scatter (FSC) and Side Scatter (SSC) parameters.
   4. Select linear scale as a Hoechst parameter by unchecking the log scale, and check the Width (W) of the voltage pulse for FCS, SSC, and Hoechst.
      ​NOTE: All the parameters are shown by default in logarithmic (log) scale, except for FSC and SSC which are in linear scale. All the parameters are analyzed by the Area (A) and the Height (H) of the voltage pulse.
   5. On the Global Worksheet, create a dot plot with FSC-A on the x-axis and SSC-A on the y-axis.
   6. Run the unstained spleen sample by clicking Acquire Data on the Acquisition Dashboard.
   7. Set the appropriate FSC and SSC settings to visualize the cells by modifying the voltage values in the Parameters section, and create a gate to select all the cells displayed in the FSC-A/SSC-A dot plot by clicking on Polygon Gate on the workspace toolbar of the Global Worksheet.
   8. Display the gated cells in histograms with each fluorescence parameter on the x-axis.
   9. Run unstained and fully stained spleen samples to adjust the fluorescence detector (PMT) to have a clear separation between negative and positive signals of the stained cells for each fluorescence parameter.
   10. To perform compensation setup, click on Experiment in the workspace ribbon, and under the Compensation Setup section, select Create Compensation Controls. Uncheck Include Unstained Control Tube/Well and click OK.
       ​NOTE: This operation will result in the creation of a specimen named Compensation Controls and a Normal Worksheet containing several sheets corresponding to each selected parameter.
   11. Run a sample of compensation beads (see section 6); set the appropriate FSC and SSC settings to visualize the beads by modifying the voltage values and the acquisition threshold of 5,000 on FSC parameters in the Cytometer window.
   12. Adjust the P1 gate on the bead population, and check that the positive and negative peaks are both visible on the x-axis. Repeat this operation for each compensation bead sample, and finally record each sample file by clicking Record Data on the Acquisition Dashboard (record at least 5,000 events for each sample).
   13. For each recorded bead sample, set the P2 and P3 gates on the positive and negative peaks, respectively.
   14. Run the cell samples for compensation (see steps 4.2 and 4.4.7, and sections 5 and 6). Modify the FSC and SSC voltages and the threshold value to visualize the cells, adjust the P1 gate, and finally record each sample file (record at least 10,000 events). Set the P2 and P3 gates on the positive and negative peaks, respectively.
       NOTE: For the compensation of the Hoechst channel, use the G₀/G₁ as the negative peak (P3) and the G₂/M as the positive one (P2).
   15. Click on Experiment in the workspace ribbon section and in the Compensation Setup section, select Calculate Compensation.
   16. Name the created compensation setting, link, and save it to the current experiment.
2. Experimental sample acquisition
   1. Open a specimen by clicking New Specimen on the browser toolbar, and create the gating strategy in the Global Worksheet.
      NOTE: The gating strategy of sample acquisition is similar to that of sample analysis, described in Figure 2 and Section 9.
   2. Display All Event populations in a histogram with CD3-A on the x-axis. Create an Interval Gate to select only the CD3⁺ cells.
   3. On the Acquisition Dashboard, select storage gate as All Events for LN samples, and either All Events or CD3⁺ cells for spleen samples.
   4. Run the experimental samples at low speed, and finally record all the files making sure to collect at least 100-200 antigen-specific CD8 T cells for each sample from the vaccinated mice.
      NOTE: The file size of experimental samples is usually big (30-120 MB), especially when the frequency of antigen-specific CD8 T cells is low. Hence, high numbers of events (> 1 × 10⁶) have to be collected to record at least 100-200 antigen-specific CD8 T cells. Big files might slow down the subsequent data analysis process. The acquisition of only CD3⁺ cells in spleen samples (see step 8.2.2 above) is helpful to keep the file size smaller.
   5. Run and record the positive control for cell cycle analysis, i.e., BM sample from untreated mice.

9. Data Analysis

1. Open the software (see Table of Materials), and create different groups corresponding to the different organs to be analyzed by clicking Create Group in the workspace ribbon section (i.e., create group "a-LNs"; "b-spleen"; "c-BM").
   NOTE: Newly created groups will appear in the group list, while the "Compensation" group is automatically generated by the software.
2. Open the Modify Group window by double-clicking on the group name, and check that the newly created groups are synchronized. If not, insert a checkmark on the function Synchronized.
3. Drag each .fcs file in its corresponding group.
4. Create the gating strategy starting with the "a-LNs" group.
5. Double-click on the fully stained sample in the group to open the graph window; the x-and y-axis are labeled as in the fcs files (see flow cytometer settings, Table 2).
6. Display the total events acquired for this sample in a dot plot with DNA-A on the x-axis and DNA-W on the y-axis.
7. Select only the single cell population by clicking on Rectangle in the gating tool section of the graph window.
   NOTE: Single cells have DNA-A values as follows: 2N (low): between 2N and 4N (intermediate), or equal to 4N (high), while DNA-W values are identical for all of them (step 1 of Figure 2).
8. Double-click in the center of the rectangular gate to display single cells in a dot plot with the FSC-A parameter on the x-axis and dead cell dye on the y-axis.
9. Select only the live cell population by clicking on Polygon in the gating tool section of the graph window. Live cells are negative for the dead cell dye (step 2 of Figure 2).
10. Double click in the center of the polygonal gate to display the cells in a dot plot with the FSC-A parameter on the x-axis and the SSC-A parameter on the y-axis.
11. Click on Rectangle, and create a "relaxed" gate to include all the single live cells in that graph(step 3 of Figure 2).
12. Double-click in the center of the "relaxed" gate to display the cells in a dot plot with CD3 on the x-axis and CD8 on the y-axis.
13. Select the CD3⁺CD8⁺ cells by clicking on Polygon (step 4 of Figure 2).
14. Double-click in the center of the CD3⁺CD8⁺ gate to display the cells in a dot plot with Tetr-gag on the x-axis and Pent-gag on the y-axis.
15. Select the antigen-specific CD8 T cells (positive for both Tetr-gag and Pent-gag) by clicking on Polygon (step 5 of Figure 2).
16. Double click in the center of the gag-specific gate to display the cells in a dot plot with DNA-A on the x-axis and Ki67 on the y-axis (Figure 3).
17. Select the cells in the different cell cycle phases by clicking on Quad in the gating tool section of the graph window.
    NOTE: Cells in the G₀ phase are Ki67neg-DNA low cells (bottom left quadrant); cells in G₁ are Ki67pos-DNA low (upper left quadrant); cells in S-G2/M are Ki67pos-DNA intermediate/high (top right quadrant) (Figure 3).
18. Copy the gating strategy created in one sample to the corresponding group to apply the gates to all the samples of the group.
19. Repeat steps 9.5 to 9.18 for the "a-LN group".
20. Check that all the gates are appropriate for each sample of the "b-spleen" group. To analyze the cell cycle among the BM cells (positive control), click in the center of the "relaxed" gate to display the cells in a dot plot with DNA-A on the x-axis and Ki67 on the y-axis.
21. Check that all the gates are appropriate for each sample of the 3 groups (i.e., for cells from spleen, LN, and BM).
    NOTE: Single cell population gate (step 9.7) and Quad gate for cell cycle (step 9.17) may have different gate coordinates in different samples, mainly due to the possible slight differences of the Hoechst dye intensity between samples (section 6.2). For this reason, it might be necessary to modify the Single-cell population gate and the Quad gates for the cell cycle in each sample. This will be done as follows: double click on the group name, and remove the synchronization from the group properties. This operation allows the modification of the gates in one sample without modifying the same gates in all the other samples of the group. After synchronization removal, modify the gates where necessary.
22. To visualize the results obtained by this analysis, click on Layout Editor in the workspace ribbon section to open it. Drag each gate of the gating strategy in the sample pane to the layout editor, and place the plots according to the sequence of the gating strategy. If necessary, change the graph type by double-clicking on the corresponding plot in the layout and selecting the appropriate Type in the Graph Definition window.
23. Click on the Group and iterate by functions on the layout ribbon to visualize the results obtained in each organ, and compare different samples.

Table 1: Flow cytometer settings.

Table 2: Flow cytometer settings.