Analysis of Immune Synapses between Human T Cells and SEB-Loaded Raji Cells using Imaging Flow Cytometry

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of Pan-leukocytes

1. Draw 1 mL of peripheral blood from a healthy donor (or patient) in a heparinized syringe. Make sure to have approval by the responsible ethics committee for the blood donation.
2. Mix 1 mL of human peripheral blood with 30 mL of ACK lysis buffer (150 mM NH₄Cl, 1 mM KHCO₃, and 0.1 mM EDTA, pH 7.0) in a 50 mL tube and incubate for 8 min at room temperature.
3. Fill the tubes with PBS and centrifuge at 300 x g for 6 min. Aspirate the supernatant and resuspend the pellet in 30 mL of ACK lysis buffer.
4. Repeat steps 1.2 and 1.3 until the supernatant is clear. Finally, wash the cells in PBS, centrifuge at 300 x g for 6 min at room temperature, and resuspend the cell pellet in 2 mL of culture medium (RPMI1640 + 10% FCS). Incubate the cells at 37 °C for 60 min.

2. Loading of Raji Cells with SEB

1. Prepare two 15 mL Falcon tubes with 1.5 x 10⁶ Raji cells per tube. Spin down the cells (300 x g for 6 min at room temperature) and discard the supernatant.
2. Resuspend the cells in residual medium (about 50–100 µL), add 1.9 µL (1.9 µG) SEB, and incubate at room temperature for 15 min. Add 5 mL of culture medium, spin down the cells (300 x g for 6 min at room temperature), and resuspend the pellet in culture medium (RPMI1640 + 10% FCS) at a density of 1 x 10⁶ cells/mL.

3. Induction of Immune Synapses and Staining Protocol

1. Pipette 500 µL of the preparation of SEB-loaded Raji cells into a FACS tube and 500 µL of the preparation of unloaded Raji cells into another FACS tube. Add 650 µL of pan-leukocytes to each tube and spin down the cells (300 x g for 10 min at room temperature). Discard the supernatant and resuspend the pellet in 150 µL of culture medium (RPMI1640 + 10% FCS). Incubate at 37 °C (typically for 45 min).
2. Gently vortex the cells (10 s at 1,000 rpm) and add 1.5 mL of paraformaldehyde (1.5%) during the vortex to fix the cells. Stop the fixation by adding 1 mL of PBS + 1% BSA. Pellet the cells (300 x g for 10 min at room temperature and resuspension the cell pellet in 1 mL of PBS + 1% BSA after incubating at room temperature for 10 min.
3. Pellet (300 x g for 10 min at room temperature) and resuspend the cells in 100 µL of PBS + 1% BSA + 0.1% saponin for 15 min at room temperature to permeabilize the cells (96-well plate, U-shaped).
4. Wash the cells in PBS + 1% BSA + 0.1% saponin with centrifugation (300 x g for 10 min at room temperature) and resuspend the cell pellet in 50 µL of PBS + 1% BSA + 0.1% saponin containing fluorophore-labeled antibodies or compounds (CD3-PE-TxRed (1:30), Phalloidin-AF647 (1:150), and DAPI (1:3,000)).
5. Incubate the cells at room temperature in the dark for 30 min. Wash the cells 3 times by adding 1 mL of PBS + 1% BSA + 0.1% saponin. Centrifuge at 300 x g for 10 min at room temperature. Re-resuspend the cells in 60 µL of PBS for imaging flow cytometry.

4. Image Acquisition Using a Flow Cytometer

NOTE: The following image acquisition procedure and data analysis are based on imaging flow cytometry using software such as imagestream (IS100), INSPIRE, and IDEAS. However, other flow cytometers and analysis software can also be used.

1. Open the analysis software on the computer connected to the imaging flow cytometer and click on Initialize Fluidics in the Instrument menu. Apply the beads on the right port when prompted to do so.
2. Load the default template from the File menu and click on Run/Setup. Choose Beads from the View dropdown menu.
3. Adjust the bright-field illuminator by clicking on Set Intensity if the indicated value is below 200.
4. Run the calibration and test routine in the Assist tab by clicking on Start All.
5. Click on Flush/Lock/Load and apply the samples in the left port when prompted to do so. After loading the cells in the flow cytometer, open the Cell Classifier and adjust the values as follows: peak intensity upper limit at 1,022 for each channel, peak intensity lower limit at 50 for channel 2 (DAPI) and channel 5 (CD3-Pe-TxR), area lower limit at 50 for channel 1 (side scatter), and upper limit at 1,500.
6. Change the excitation laser power to 405 nm (15 mW), 488 nm (200 mW), and 647 nm (90 mW) in the Setup tab.
7. Switch the View dropdown menu between Cells and Beads to evaluate the cell classifier and laser power adjustments.
   NOTE: Make sure that all cells and cell couples are found in the Cell View and that cell clumps, debris, and images with saturated pixels are found in the Debris View by changing the cell classifiers and/or the excitation laser powers.
8. Define the sample name and the number of images to acquire (15,000–25,000 for samples and 500 for compensation controls) in the Setup tab. Click on Run/Setup to start the acquisition.