PBMC/T-cells were stimulated with 1 μg/mL purified plate-bound anti-CD3 mAb (clone Hit-3a; BioLegend) and soluble 0.3 μg/mL anti-CD28 mAb (clone CD28.2; eBioscence) for 18 hours in complete RPMI medium in 96 flat-bottom wells with a starting concentration of 2 × 105 cells/well (1 × 106/mL) of PBMCs or 1 × 105 cells/well (1 × 106/mL) of T-cells at 37° C, 5% CO2. For neutrophil/T-cell suppression assay, neutrophils were cocultured with CD3+ PBMC/T-cells at a 3 : 1 ratio at 37° C, 5% CO2.
CD25 surface expression was assessed using flow cytometry. The following mAbs were used for PBMC/T-cell staining: CD4-PE-Cy7 (clone SFCI12T4D11), CD8-APC A750 (clone B9.11) (Beckman Coulter), CD3-APC (clone SK7) (BD Biosciences), and CD25-PerCP-Cy5.5 (clone BC96) (BioLegend).
To detect IFN-γ by intracellular staining, brefeldin (10 μg/mL) was added for the last 4 hours of incubation. The cells were washed, fixed, permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer´s protocol, and stained with IFN-γ-BV421 (clone 4S.B3) (BioLegend).
CD25 surface expression was assessed using flow cytometry. The following mAbs were used for PBMC/T-cell staining: CD4-PE-Cy7 (clone SFCI12T4D11), CD8-APC A750 (clone B9.11) (Beckman Coulter), CD3-APC (clone SK7) (BD Biosciences), and CD25-PerCP-Cy5.5 (clone BC96) (BioLegend).
To detect IFN-γ by intracellular staining, brefeldin (10 μg/mL) was added for the last 4 hours of incubation. The cells were washed, fixed, permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer´s protocol, and stained with IFN-γ-BV421 (clone 4S.B3) (BioLegend).