Flow Cytometry Analysis of Th17 and Treg Cells

Flow cytometry was performed as described elsewhere [28 (link)–29 (link)]. In brief, for Th17 detection, approximately 10⁶ splenocytes were prepared and incubated with PMA (20 ng/m) and ionomycin (1 μg/mL) in the presence of monensin (2 mmol/ml, Sigma-Aldrich, St. Louis, MO, USA) for 4 h (37°C, 5% CO₂). The cells were subjected to allophycocyanin (APC)-anti-CD3 and phycoreythrin (PE)-Cy5-anti-CD4 staining at 4°C for 30 minutes in the dark. After surface staining, the cells were fixed in paraformaldehyde and permeabilized in Perm/Fix solution according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA) and then intracellularly stained with anti-mouse PE-IL-17. To detect Treg cells, the cell suspensions were stained with APC-anti-CD3, PE-cy5-anti-CD4, and fluorescein isothiocyanate (FITC)-anti-CD25 at 4°C for 30 minutes; treated with eBioscience Perm/Fix mixture; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). All of the stained cells were evaluated using a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using Diva software (BD Biosciences).