40592 v08h2 b

In order to detect SARS-CoV-2-specific B cells, biotin-labeled SARS-CoV-2 spike RBD protein (40592-V08H2-B; Sino Biological, Beijing, China) was mixed with Streptavidin BV421-11 (405225; Biolegend, California, CA, USA) at a molar ratio of 4:1 for 1 h to obtain an antigen probe. According to the manufacturer's instructions, peripheral blood mononuclear cells were isolated from whole heparinized blood by Histopaque (10771; Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation. After washing with flow cytometry staining (FACS) buffer (phosphate-buffered saline with 2% fetal bovine serum), staining was performed for 30 min at 4°C with an antigen probe (1:33.3) and the following binding antibodies at 1:50 dilution: anti-human CD3 (300430), anti-human CD19 (302212), anti-human CD21 (354918), and anti-human CD27 (356406) all purchased from Biolegend. After staining, the cells were re-washed and suspended in 200 µL FACS buffer. Samples were evaluated using a CytoFLEX cytometer (Beckman Coulter, Inc., Brea, CA, USA), and FlowJo software version 10.0.7r2 (Treestar Inc., Ashland, OR, USA) was used for data analysis.

The stained peripheral blood mononuclear cells were tested by Beckman flow cytometry (Beckman Coulter, Inc., California, USA). The specific steps were as follows: first, we mixed the biotinylated SARS-CoV-2 spike RBD protein (40592-V08H2-B, Sino biological, Beijing, China) with streptavidin-BV421 (405225, Biolegend, California, USA) in a 4:1 mole ratio and leave for 1 h to get the antigen probe; second, peripheral blood mononuclear cells were obtained from whole heparinized blood; the density gradient centrifugation was performed by Histopaque (10771, Sigma–Aldrich, St Louis, Missouri, USA) and then cleaned by cytometric staining buffer (FACS, 2%FBS) cells, besides we added antigen probe (1:33.3), fluorescence-coupled antibody [anti-human IgG Fc (410722, Biolegend), anti-human IgM (314524, Anti-human) CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), and anti-human CD27 (356406, Biolegend)] into the cells and dyed at 4°C for 30 min under dark condition. After being re-suspended with FACS buffer, the samples were tested on the machine. The data were analyzed by Flow Jo software (V10.0.7).