- Thaw PBMC following our standard procedure and label the cells with CFSE. We have a separate protocol for CFSE labeling.
- 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.