Fitc anti human cd19

The stored Human peripheral blood mononuclear cells (PBMCs) were first treated with following steps. The PBMCs were quickly thawed in a 37 °C water bath. The suspensions were centrifuged at 500 × g for 5 min to remove the supernatant. The PBMCs were resuspended with 2% FACS buffer (phosphate buffered saline containing 2% fetal bovine serum). The PBMCs washed with 2% FACS buffer were filtered by Corning (Falcon) 100 μm cell strainers. After the pretreatment, the PBMCs were incubated with human Fc block (BioLegend) in the dark at 4 °C for 15 min. The PBMCs were then incubated with APC-cy7 anti-human CD3 (BioLegend), APC anti-human CD20 (BioLegend), FITC anti-human CD19 (BioLegend) and spike (S) protein-biotin in the dark at 4 °C for 30 min. The cells were then incubated with PerCP/Cyanine5.5 Streptavidin (BioLegend) and PE Streptavidin (BioLegend) in the dark at 4 °C for 30 min.

LCLs were stained by multicolor flow cytometry with FITC anti-human CD19; APC-CY7 anti-human CD27; PerCP anti-human CD38; and APC anti-human CD24 (Biolegend). BD CompBeads (Becton and Dickinson) were used for compensation according to the manufacturer's instructions. Unstained cells were used to exclude background fluorescence and isotype controls to determine antibody specificity. LCL viability was monitored using 7-amino-actinomycin D (eBiosciences, USA). Data reading and acquisition was performed using the Flow Cytometer Cyan ADP (Beckman Coulter). FlowJo Software (TreeStar) was used for data analysis.

The blood samples were centrifuged to collect the plasma. White blood cells were obtained after red blood cells were lysed and centrifuged at 1000 rpm. After incubating with Fc receptor blocking solution (Human TruStain FcX™, Biolegend, California, USA) for 30 min., the cells were further incubated with FITC anti-human CD19, APC anti-human CD4 and PE anti-human CD14 (Biolegend, California, USA) for 30 min. Flow cytometry analysis was done in FACS buffer containing PBS plus 2% FBS using BD LSRFortessa instrument. FlowJo v10.2 software (Treestar) was used for data analysis. Cells were sorted using BD FACSAria.