Lymphocyte Immunophenotyping Protocol

Cell surface and intracellular markers expression was evaluated on the fresh whole EDTA-anti-coagulated peripheral blood according to the method described in a previous study [20 (link)] conducted in Sweden. The lymphocytes were isolated using density gradient centrifugation at 900×g at room temperature for 20 min over Lymphoprep™ (Nycomed, Oslo, Norway) according to the manufacturer’s instructions. Briefly, for the surface staining, the lymphocytes were incubated using FITC-conjugated anti-human CD4⁺ antibody (BD Biosciences, San Jose, CA, USA), PE-conjugated anti-human CD25⁺ antibody (BD Biosciences), PerCP-labeled anti-human CD127 antibody (BD Biosciences), APC anti-human CD3⁺ monoclonal antibody (BD Biosciences), and eBioscienceTM Anti-Human Foxp3 Staining St FITC (Cat. No. 71-5776, Thermo Fisher Scientific, Hudson, NH, USA) for 20 min at 4°C in the dark. Then, the lymphocytes were centrifuged (1200 r/min) for 5 min and re-suspended in 2 ml PBS. The above samples were analyzed using an FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). All flow cytometry data were analyzed using BD DIVA software 4.1 (BD Biosciences, San Jose, CA, USA). The results are presented as the percentage of CD4⁺CD25⁺ T cells, Treg CD127⁺ T cells, or Treg Foxp3⁺ cells of total lymphocytes.