Brefeldin-A (Sigma Aldrich; cat.: B7651; 1 mg/mL) was added during the last 4 h of culture to impair protein secretion, allowing for intracellular cytokine staining. A Human FOXP3 buffer set (BD Pharmigen; cat.: 560098) was used for FOXP3 tagging according to the manufacturer’s instructions. Cells were incubated at 5% CO2 at 37 °C for 4 h. Next, to follow the ex vivo protocol, the plaque was centrifugated (8 min, 244× g, 4 °C), and the supernatant was removed. Extracellular conjugated antibodies anti-CD4 (APCCy7; clone: RPA-T4), anti-CD25 (PECy7; clone: BC96), anti-CD39 (PE; clone: TU66), and anti-CD73 (APC; clone: AD2) (BD Pharmigen, San Diego, CA, USA/Ebioscience, San Diego, CA, USA) and IgG isotypes control antibodies IgG3-FITC (clone J606, mouse BALB/c IgG3, κ, cat. 555578) and IgG1-PE-Cy-7 (clone O4–46, mouse IgG1, κ, cat. 561316), were added at the volume suggested by the manufacturer. Cells were incubated for 15 min at 4 °C and later washed with 150 μL of PBS/well. After centrifugation (8 min, 577× g, 4 °C) and supernatant removal, extracellular staining was fixed using 100 μL of formaldehyde 4% (Sigma Aldrich; cat.: 252549) diluted in 100 μL of PBS and incubated at room temperature (25 °C) for 20 min. Following centrifugation (8 min, 577× g, 4 °C) the supernatant was discarded and samples were washed with 150 μL/well of PBS. Again, the plaque was centrifugated (8 min, 577× g, 4 °C) and the supernatant was discarded. To perform intracellular staining, cells were permeabilized with 150 μL of permeabilization buffer (PBS + BSA (0.5%) + Saponin A (Sigma Aldrich; cat.: S7900) (0.5%)) for 10 min at room temperature (25 °C). After centrifugation (8 min, 577× g, 4 °C) and removal of the supernatant, intracellular antibodies anti-FOXP3 (Alexa Fluor 488; clone: 259D/C7) and anti-IL-10 (APC; clone: JES3–19F1) (BD Pharmigen, San Diego, CA, USA/Ebioscience, San Diego, CA, USA) were added at a volume suggested by the manufacturer (BD Bioscience, CA, USA). Then, the plate was incubated for 30 min at room temperature (25 °C) and after this 150 μL/well of permeabilization buffer was added. Following centrifugation (8 min, 577× g, 4 °C), the supernatant was removed. Finally, 200 μL/well of Wash B (PBS/BSA) was added and the samples were transferred to FACS tubes and stored at 4 °C. At least 50,000 gated events were acquired using FACS CANTO II (BD Biosciences, USA) and analyzed using FlowJo software version 10.4 (BD, Ashland, OR, USA) [19].