Anti cd14 pacific blue

Briefly, cryopreserved PBMC were thawed in pre-warmed RPMI 1640 (Cat. 12019003, Corning, USA) with 10% fetal bovine serum and 50 IU/ml benzonase (Sigma, USA). Thereafter, PBMCs were washed, surface stained with antibody cocktail containing following antibodies: anti-CD3-Alexflour 700, anti-CD8-Pacific Blue, anti-CD14-Pacific Blue, anti-CD19-PECy7, anti-CD27-APCCy7, anti-IgG-FITC, anti-IgM-PECy5 (above antibodies are all from BD Biosciences), anti-CD20-ECD (Beckman Coulter) and anti-RBD-PE in a total volume of 50 μL on ice in dark for one hour, followed by Live/Dead staining with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Cat. L34957, Invitrogen, USA). The cells were washed and suspended in cold PBS with 2mM EDTA. The stained PBMC were filtered by 70-μm cell mesh (Cat. 352235, Corning, USA) and sorted using a five-laser FACS Aria cell sorter III driven by FACS Diva software. Single cells with the phenotype of CD3-, CD8-, DAPI-, CD14- CD19+, CD20+, CD27+, IgM-, IgG+, RBD+ were defined as SARS-CoV-2 RBD specific memory B cells. Single cells were sorted into 96-well PCR plates containing 20 μL of cell lysis buffer per well under yield mode, and PCR plates were quickly frozen on dry ice and stored at -80˚C overnight. Then RT-PCR were performed to amplify the variable regions of the heavy chain (V_H) and light Chain (V_L) as previously reported (27 (link)).