Comprehensive Immune Cell Profiling by FCM

FCM assay was conducted as we previously reported with several modifications [2 (link), 15 (link)]. In details, the unstimulated MNCs (day 0) and the MNC-derived cells (day 7, day10, day 14) were harvested by centrifugation at 1200 rpm for 5 min at room temperature (RT) and washed by 1 × PBS for twice. Then, the cells were resuspended in phosphate buffer solution (PBS) contained 2% fetal bovine serum (FBS) and labelled with the fluorescence conjugated antibodies including anti-CD3-PE, anti-CD3-FITC, anti-CD4-PE, anti-CD8-PE-Cy7, anti-CD56-APC, anti-CD16-FITC, anti-NKG2D-Percp5.5, anti-NKp44-APC-Cy7, anti-NKp46-PE-Cy7, anti-NKG2A-PE, anti-CD107a-PE, 7-AAD, PI or Annexin V-FITC (BD Biosci, USA) in dark for 30 min. Finally, the cells were washed by 1 × PBS for twice and turned to FACS Canto II (BD Biosci, USA) flow cytometer for detection and FlowJo 10.0 software (Tree Star, USA) for analysis. The detailed information of the indicated antibodies was listed in Additional file 1: Table S2.