Anti igg pecy7

PBMC were isolated via a standard Ficoll gradient. Informed consent in writing was obtained from each donor. B cells were enriched from PBMC using Dynabeads Untouched Human B cells Kit (Thermo Fisher). For further analysis, B cells were incubated with human Fc block (BD Biosciences) for 10’ at RT, followed by biotinylated HBsAg for 30’ on ice (HBsAg by Roche Diagnostics; EZ-Link Sulfo-NHS-Biotinylation kit, Thermo Fisher). Memory B cell staining: 30’ on ice in PBS+0.5% BSA with live-dead stain (Thermo Fisher), anti-CD19-eF450 (eBioscience), anti-IgG-Pe-Cy7 (BD) and Streptavidin-APC (eBioscience). Cells were analyzed via flow cytometry on Cytoflex S (Beckman Coulter) or sorted with MoFlo II (Beckman Coulter) into lysis buffer, i.e. PBS+12U RNasin (Promega), 100 mM DTT (Thermo Fisher). PCR plates were sealed and stored at -80°C. IgG gene identification via PCR and sequencing from single B cells was performed as described elsewhere (16 (link)).

Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody as previously described (Graham et al., 2021 (link)). Sorting baits (SARS-CoV-2 Spike and RBD) was pre-complexed with the streptavidin fluorophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362) or RBD-Alexa488 and RBD-APC. Live CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺Spike⁺Spike⁺ or CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺RBD⁺RBD⁺ cells were sorted using a BD FACS Melody into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers (Bioline Reagents Ltd) following the manufacturer’s protocol.