FoxP3 staining

Following surface staining and prior to fixation, PBMC were washed and resuspended in 1 ml of freshly prepared FoxP3 fixation/permeabilization working solution, consisting of FoxP3 fixation/permeabilization concentrate (eBioscience, Hatfield, UK) diluted with FoxP3 fixation/permeabilization diluent (eBioscience) at a concentration of 1 : 3, and incubated in the dark for 30 min at +4°C. The mixture was washed in permeabilization working solution (eBioscience), resuspended in the same solution and 5 μl phycoerythrin (PE)‐labelled anti‐FoxP3 was added and incubated in the dark at room temperature for 30 min. After two wash steps the cells were resuspended in 200 μl of diluted stabilizing fixative and stored at 4°C until acquisition.