Percoll plus density gradient media

All blood samples were collected in sodium heparin tube and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation using Percoll PLUS density gradient media (GE Healthcare, Philadelphia, Pennsylvania, PA, USA) at 1500 rpm for 30 min at room temperature. PBMCs were washed twice with Hank’s balanced salt solution (HBSS) (GIBCO, New York, NY, USA) with 5% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin. Total lymphocytes were counted and stored at liquid nitrogen before analysis.
Cryopreserved PBMCs from each patient were defrosted and added in complete in RPMI1640 Medium (GIBCO, New York, NY, USA) supplement with 10% FBS. At least 200000 cells were stained with combinations of antibodies including anti-CD19-PE, anti-CD38-FITC, anti-IgD-PerCP-Cy5.5, anti-CD27 Alexa Fluor 700, anti-CD24 PE-CyTM7 (BD Biosciences, San Jose, CA, USA), anti-CD268 (BAFFR)-APC, anti-CD267 (TACI)-APC and anti-CD269 (BCMA)-APC (BioLegend, San Diego, CA, USA). The cells were re-suspended in the buffer before performed flow cytometry using BD LSR II Flow Cytometry Analyzer (BD Biosciences, San Jose, CA, USA). Flow cytometry data were analyzed with Flowjo software version 10 (Tree star, Ashland, Oregon, USA).