Multiparametric Flow Cytometry Analysis of Immune Cell Populations

FCM analysis was performed as we recently reported [20 (link), 22 (link)]. In brief, the fresh-enriched MNCs (day 0) and the MNC-derived cells (day 14) were harvested by centrifugation at 300×g for 5 min and resuspended by 1 × PBS (Solarbio, China) for twice. After that, the cells were incubated in 1 × PBS (Solarbio, China) with 2% fetal bovine serum (FBS) (Australia) and the fluorescence-conjugated antibodies such as anti-CD3-PE (BioLegend, USA), anti-CD3-APC (BioLegend, USA), anti-CD4-PE (BioLegend, USA), anti-CD8-PE-Cy7 (BioLegend, USA), anti-CD56-APC (BioLegend, USA), anti-CD16-FITC (BioLegend, USA), anti-CD25-FITC (BioLegend, USA), anti-NKG2D-perCP-Cy5.5 (BioLegend, USA), anti-NKp44-APC-Cy7, anti-NKp46-PE-Cy7 (BD Biosci, USA), anti-NKG2A-PE (BD Biosci, USA), anti-CD107a-PE (BD Biosci, USA), 7-AAD (BD Pharmigen), Propidium iodide (PI) (BD Pharmigen, USA) or Annexin V-FITC (Tianjin Sungene Biotech, China) in dark for 30 min. Finally, the cells were washed and turned to FACS Canto II (BD Biosci, USA) and FlowJo 10.0 software (Tree Star, USA) for analysis. The list of the indicated antibodies was available in Additional file 1: Table S3.