Anti foxp3 percp

All antibodies used for cell labeling were purchased from eBioscience. For intracellular cytokine measurement, MLN cells were stimulated for 5 h with PMA (1 μg/mL, Sigma Aldrich) and ionomycin (50 μg/mL, BD Biosciences) in the presence of monensin (0.1 mg/mL, Sigma Aldrich) and placed in a 37 °C and 5% CO₂. MLN cells were washed with PBS and surface-labeled with anti-CD4 –FITC (Biolegend, Uithoorn, Netherlands) and anti CD25-PE (BD Biosciences). MLN cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) and stained intra cellularly with anti-IL-17-PE (Biolegend, Uithoorn, Netherlands) and anti-FoxP3-PerCP (BD Biosciences). The stained cells were analyzed using FACS Calibur, and the data were analyzed using Cell Quest Pro software.

The PBMCs were stained for CD4, PD-1 and CTLA-4, and then measured using flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA). We discriminated the lymphocyte population using forward scatter and side scatter. Within groups, we gated the subgroups of CD4⁺ T lymphocytes and measured the expressions of PD-1, and CTLA-4. The gating cut-off values were based on isotype staining. We also stained the PBMCs for CD4, CD25, and Foxp3, and defined positive staining for all three as showing Treg cells in the lymphocyte population. We measured CD3-/CD14-/HLA-DR- cells and then gated CD11b⁺/CD33⁺ cells to represent MDSCs using a modified protocol [18 (link)].
The staining antibodies were anti-CD4-APC, anti-CD25-FITC, anti-Foxp3-PerCP antibodies (BD Biosciences, San Jose, CA, USA), anti-PD-1-PE, and anti-CTLA-4-PEcy7.0 (eBiosciences, San Diego, CA, USA). Data were analyzed using BD FACSuite V software (BD Biosciences, San Jose, CA, USA).