Facs fixing buffer

At 13 hrs, cells were harvested by EDTA treatment and washed once using 1 ml ice-cold FACS buffer (2% FCS in PBS). PE conjugated monoclonal antibody 25.D1-16 was then added at a final concentration of 0.6 μg/ml. Cells were incubated for 30 min on ice in the dark and washed three times using 1 ml FACS buffer, re-suspended in 0.2 ml FACS fixing buffer (BD) and analyzed by flow cytometry using FlowJo software (ThreeStar, San Carlos, CA, USA).

PBMCs were washed once with 1 ml ice-cold FACS buffer (2% FCS in PBS) and the cell density was adjusted to 1 × 10⁶ cells/100 μl. APC-conjugated anti-human CD3 monoclonal antibody (BD, USA), FITC-conjugated anti-human CD8 (BD, USA), PE-conjugated anti-human CD279 monoclonal antibody (BD, USA) and FVS620 (BD, USA) were added and incubated in the dark for 15 min at room temperature. For FoxP3 staining, PBMCs were first stained with FITC-conjugated anti-Human CD4 monoclonal antibody (BD, USA), APC-conjugated anti-Human CD25 monoclonal antibody (BD, USA) and FVS620 (BD, USA) for 15 min in the dark at room temperature. After fixation and permeabilization, the cells were stained with PE-conjugated anti-human FoxP3 monoclonal antibody (BD, USA) overnight at 4°C. The cells were then washed three times with 1 ml of FACS buffer, resuspended in 0.5 ml of FACS fixing buffer (BD, USA) and acquired using CytoFLEX S flow cytometer (Beckman, USA). The data was analyzed by FlowJo software version VX (ThreeStar, San Carlos, CA, USA).

0.6 × 10⁶ PBMCs were washed with phosphate-buffered saline (PBS) (Sigma) and first stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Life Technologies) for 20 min, followed by surface staining with mouse anti-human CD3-AF700 antibody, mouse anti-human CD8-APC-H7 antibody, mouse anti-human CD4-FITC antibody, mouse anti-human TIGIT-PE-A antibody, mouse anti-human TIM-3-BB515 antibody and 2B4-APC antibody, CTLA4, PD-1, and KLRG1 (BD) for 20 min. Finally, 300 µL FACS fixing buffer (BD) was added to fix the cells for acquisition by flow cytometry. All data were acquired on BD LSRFortessa™ Cell Analyzer (BD).