The expression of CD39 and CD73 in circulating CD4+CD25+Foxp3+ T cells, as Tregs, was evaluated using the Human Regulatory T cell Staining Kit (eBioscience, San Diego, CA, USA), according to the manufacturer’s protocol. Single-cell suspensions were incubated with PerCP-conjugated anti-human CD4 (BD Biosciences), allophycocyanin (APC)-conjugated anti-human CD25 (BD Biosciences), FITC-conjugated anti-human CD39 (BD Biosciences), and PE-cy7-conjugated anti-human CD73 (BD Biosciences) antibodies for 30 min in the dark at 4 °C to stain surface-expressed factors.
After washing with flow cytometry staining buffer, the PBMCs were incubated with 1 ml freshly prepared Foxp3 fixation/permeabilization buffer for 20 min at 4 °C in the dark. Then, the cells were washed twice with 2 ml freshly prepared 1 × permeabilization buffer. Next, the cells were stained using a PE-conjugated anti-human Foxp3 (eBioscience) antibody for 30 min in the dark at 4 °C. After washing twice, the number of Foxp3-positive cells in the CD4+CD25+ cell gating was evaluated by flow cytometry, and the frequency of Foxp3+ Treg cells was expressed as a percentage of the total CD4+ CD25+ cells.
After washing with flow cytometry staining buffer, the PBMCs were incubated with 1 ml freshly prepared Foxp3 fixation/permeabilization buffer for 20 min at 4 °C in the dark. Then, the cells were washed twice with 2 ml freshly prepared 1 × permeabilization buffer. Next, the cells were stained using a PE-conjugated anti-human Foxp3 (eBioscience) antibody for 30 min in the dark at 4 °C. After washing twice, the number of Foxp3-positive cells in the CD4+CD25+ cell gating was evaluated by flow cytometry, and the frequency of Foxp3+ Treg cells was expressed as a percentage of the total CD4+ CD25+ cells.