Thaw Tregs and adjust cell concentration to 106 per mL in culture medium.
Prepare 2-fold dilutions of the Tregs in medium to test a range of Treg to effector (labeled PBMC) ratios.
Add Tregs to a U bottom 96 well plate at 100 uL per well.
After labeling, adjust the PBMC to 5 x 106 cells per mL and add 50 uL per well of a 96 well plate.
Dilute anti CD3 antibody to 4 ug/mL and add 50 uL per well of a 96 well plate.
Incubate at 37°C, 6% CO2 for 4–5 days.
Collect cells from wells and stain with anti CD8.
Analyze on a flow cytometer gating on the CD8+ cell population.
Controls should include:
labeled PBMC without anti CD3 or Tregs added and
labeled PBMC with anti CD3 but no Tregs.
The PBMC without anti CD3 will not proliferate but will provide a measure of fluorescence of cells that have not divided.
The PBMC with anti CD3 will have discrete peaks of fluorescence intensity marking each round of cell division. Five or six of these peaks should be easily seen in the plots of PBMC without Tregs added. As more Tregs are cultured with the PBMC, the number of peaks decreases and more cells remain at the highest fluorescence intensity seen in the undivided cells.
For mouse Tregs, use spleen cells rather than PBMC. The proportion of T cells in mouse spleen is lower than the proportion of T cells in PBMC. Increase the number of spleen cells per well to 500,000 to ensure sufficient CD8+ cells for analysis. The number of Tregs may also need to be increased.
We have used clone OKT3 for stimulation of human T cells and clone 145-2C11 for stimulation of mouse T cells.