Histopaque 1077 separation

For analysis of regulatory T cell frequency, peripheral blood mononuclear cells were purified from 9 mL of whole blood using Histopaque-1077 separation gradient (Sigma-Aldrich Corporation, St Louis, MO, USA) and frozen at −80 °C. After, 2 × 10⁵ cells were labeled with anti-CD4 PE Cy7 (clone SK3-BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD25 APC H7 antibodies (clone M-A251-BD Biosciences, Franklin Lakes, NJ, USA) for 30 min. Cells were permeabilized using FoxP3 buffer (BD Biosciences, Franklin Lakes, NJ, USA) and then labeled with anti-FoxP3 Alexa Fluor®488 antibody (clone 259d/ C7, BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at room temperature. The cells were analyzed by flow cytometry (FACS Canto II, BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Isotype control antibodies were used to exclude non-labeled cells specifically. For analysis of IL-10 production, peripheral blood mononuclear cells were purified and cultured with anti-CD3 and anti-CD28 antibody for 24 h, and the supernatant was collected and analyzed using CBA (BD Bioscience, Franklin Lakes, NJ, USA) by flow cytometry.