Anti cd27 apc cy7

Probe-specific single B cells were sorted as previously described [27 (link)]. Briefly, one tube of PBMCs was thawed in water at 37 °C and resuspended in a prewarmed RPMI-1640 (Corning, Corning, NY, USA) containing 10% fetal bovine serum (Sera Pro, Gremlingen, BW, Germany) and 50 IU/mL benzonase (Sigma, St. Louis, MO, USA). After washing with PBS, the PBMCs were stained with a LIVE/DEAD dye (Invitrogen, Carlsbad, CA, USA) on ice for 20 min. After washing, the PBMCs were stained with an antibody cocktail containing anti-CD3-Pacific Blue, anti-CD8-Pacific Blue, anti-CD19-BV510, anti-IgM-PercpCy5.5, anti-IgG-FITC, anti-CD27-APC-Cy7, anti-PD-1-PECy7, anti-CXCR5-APC-R700, anti-CXCR3-PECy5, anti-CD45RA-BV650, anti-CD4-BV605 (above antibodies are all from BD Biosciences), anti-CD14-eflur450 (ebioscience, USA), anti-CD20-ECD (Beckman Coulter, Bria, CA, USA) and RBD-PE. After filtering through a cell strainer (Corning, Corning, NY, USA), the PBMCs were sorted on BD FACS Aria SORP. RBD-specific single memory B cells were gated as CD3⁻, CD8⁻, DAPI⁻, CD14⁻, CD19⁺, CD20⁺, CD27⁺, IgM⁻, IgG⁺, RBD-PE⁺, and were sorted into a 96-well PCR plate containing cell lysis buffer. Then, the PCR plate was frozen immediately at −80 °C and stored overnight.

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)).