Cd10 bv510 hi10a

Biotinylated recombinant SARS-CoV-2 spike of ancestral Wuhan Hu-1 and Omicron (BA.1 or BA.2) strains were conjugated to streptavidin-allophycocyanin (APC) or streptavidin–phycoerythrin (PE) fluorophores, respectively. PBMCs were thawed and stained with Aqua viability dye (Thermo Fisher Scientific) and then surface-stained with spike probes: CD19 ECD (J3-119) (Beckman Coulter), IgA VioBlue (IS11-8E10), IgM BUV395 (G20-127), IgD PE-Cy7 (IA6-2), IgG BV786 (G18-145), CD21 BUV737 (B-ly4), CD27 BV605 (O323), streptavidin BV510 (BD Biosciences), CD14 BV510 (M5E2), CD3 BV510 (OKT3), CD8a BV510 (RPA-T8), CD16 BV510 (3G8), and CD10 BV510 (HI10a) (BioLegend). Cells were washed twice with PBS containing 1% FCS and fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSRFortessa using BD FACSDiva.

Probes for delineating SARS-CoV-2 S-specific B cells within cryopreserved human PBMC were generated by sequential addition of streptavidin-PE (Thermofisher) to trimeric S protein biotinylated using recombinant Bir-A (Avidity). SARS-CoV-2 RBD protein was directly labelled to APC using an APC Conjugation Lightning-link kit (Abcam). Cells were stained with Aqua viability dye (Thermofisher). Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), CD20 Alexa700 (2H7), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend), IgA-Vio450 (clone) (Miltenyi). Cells were washed, fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSR Fortessa or BD Aria II. Gating is shown in Supplementary Fig. 4.