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

Begin by mixing one milliliter of freshly drawn human peripheral blood with 30 milliliters of ACK lysis buffer in a 50-milliliter conical tube. After eight minutes at room temperature, fill the tube with PBS for a centrifugation wash, and resuspend the pellet in two milliliters of culture medium for a 60-minute incubation at 37 degrees Celsius.

At the end of incubation, add 5 x 10⁵ Staphylococcus aureus enterotoxin B or SEB loaded or unloaded Raji cells in 500 microliters of culture medium into individual FACS tubes, followed by 650 microliters of pan-leukocytes. Spin down the co-cultures, and resuspend the pellets in 150 microliters of fresh culture medium for a 45-minute incubation at 37 degrees Celsius.

Then, gently vortex the cells for 10 seconds at 100 RPM while adding 1.5 milliliters of 1.5% paraformaldehyde. After 10 minutes at room temperature, stop the fixation with one milliliter of PBS supplemented with 1% BSA, and collect the cells by centrifugation. Resuspend the pellets in 100 microliters of PBS plus BSA and 0.1% saponin, and add the cell samples to two individual wells of a 96-well U-bottom plate.

After 15 minutes at room temperature, centrifuge the plate and resuspend the pellets in 50 microliters of PBS plus BSA and saponin, containing the fluorophore-conjugated antibodies or compounds of interest for a 30-minute incubation in the dark at room temperature. At the end of the incubation, wash the cells three times in 150 microliters of PBS plus BSA and saponin, and resuspend the pellets in 60 microliters of PBS.

To image the cells by flow cytometry, open the analysis software, check the liquid level, and click Start Up in the instrument menu. Next, click Load, and load the samples into the port when prompted.

Under the Illumination tab, change the excitation laser power to the appropriate nanometer lengths. Then, adjust the gates for channels 4 and 11, and adjust the area for channel 1.

To evaluate the gating and laser power adjustments, switch the View menu to the respective gate, and adjust the gates and laser powers until the cells and cell couples are visible. Then, in the File Acquisition tab, define the sample name, and the number of images to acquire, and the appropriate gate for CD3 staining, and click Acquire to begin the acquisition.