Lag 3 af700

For surface staining, PBMCs from liquid nitrogen were thawed and washed twice in phosphate buffered saline containing 1% fetal bovine serum (staining buffer). Cells were incubated with directly conjugated monoclonal antibodies for 30 min at 4 °C. The cells were then washed and resuspended in staining buffer before flow cytometric analysis. The monoclonal antibodies used were anti-human CD3-PerCp-Cy5.5 or CD3-BV421, CD4-FITC or CD4-V500, CD8-APC-H7, CD45RA-PE-Cy7, CCR7-PerCp-Cy5.5 (BD Biosciences, San Diego, CA, USA), PD-1-PE, TIM-3-FITC, 2B4-APC, BTLA-BV421, CD160-PE-Cy7 (BioLegend, San Diego, CA, USA) and LAG-3-AF700 (R&D Systems, Minneapolis, MN, USA) antibodies, and corresponding isotype controls. Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences) and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).

For surface staining, peripheral blood mononuclear cells (PBMCs) were incubated with directly conjugated mAbs for 30 min at 4 °C and then washed before analysis. Antibodies used were anti-human CD3-BV786 or CD3-APC, CD4-BV711, CD8-APC-H7, CD45RA-AF700, CD95-FITC, CD25 BV510, CD73 PE-CY7, CD11b AF700, CD45 BV786, CD14 BV711, CD56-PE-CF594, CD19-FITC, HLA-DR-PE, CD160-AF488 (BD Biosciences, San Diego, CA, USA), CCR7-BV421, PD-1-PE, 2B4-APC (BioLegend, San Diego, CA, USA), TIGIT-APC (Ebioscience, San Diego, CA, USA), LAG-3-AF700 (R&D Systems, Minneapolis, MN, USA) antibodies, and corresponding isotype controls. Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).