For CD27+IgD+ B cell detection, 100 µl fresh whole blood cells were stained with APC-CY7-conjugated anti-CD19 (Biolegend, San Diego, CA, USA), APC-conjugated anti-CD27 (eBioscience, San Diego, CA, USA), and FITC-conjugated anti-IgD (eBioscience). Then the red blood cells were lysed using the RBC lysis buffer (MultiSciences, Hangzhou, China) and the left cells were analyzed on FACS Aria II. Dead cell exclusion was performed by scatter profiles and 7-AAD staining during all the flow cytometric analyses.
For CD27+IgD+ B cell sorting, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples using Ficoll density-gradient centrifugation, and then were stained as described above. After that, the aimed cells were harvested into the collection solution (RPMI 1640 supplemented with 10% FBS and 2% antibiotics) using FACS Aria II flow cytometry sorter according to the manufacturer’s instructions. The purified cells were further analyzed after sorting, the purity of which was 95–99%.
For CD27+IgD+ B cell sorting, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples using Ficoll density-gradient centrifugation, and then were stained as described above. After that, the aimed cells were harvested into the collection solution (RPMI 1640 supplemented with 10% FBS and 2% antibiotics) using FACS Aria II flow cytometry sorter according to the manufacturer’s instructions. The purified cells were further analyzed after sorting, the purity of which was 95–99%.