Igg bv786 g18 145

To generate the protein probes, the recombinant WA-1 S-2P and RBD proteins were biotinylated using the EZ-Link Micro Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions [20 (link)]. Streptavidin-conjugated fluorophores (SA-PE, SA-APC or SA-BV421) and biotinylated proteins were coupled at a 4:1 molar ratio. The cryopreserved PBMCs were thawed and stained with 100 ng of the fluorescent protein probes for 20 min at 4 °C, followed by staining with 7-aminoactinomycin D (7-AAD, Thermo Fisher, Waltham, MA, USA) and a panel of antibodies, IgM PerCP-Cy5.5 (G20-127, BD, Franklin Lakes, NJ, USA), IgD FITC (polyclonal, Southern Biotech, Birmingham, AL, USA), CD3 BV510 (SP34-2, BD), CD14 BV510 (M5E2, BioLegend, San Diego, CA, USA), CD16 BV510 (3G8, BD), CD20 BV605 (2H7, BioLegend, San Diego, CA, USA), HLA-DR BV650 (L243, BioLegend, San Diego, CA, USA) and IgG BV786 (G18-145, BD, Franklin Lakes, NJ, USA), for another 20 min at 4 °C. After staining, the cells were washed with FACS buffer (PBS supplemented with 2% heat-inactivated fetal calf serum) and fixed with 1% formaldehyde solution. The samples were acquired using a BD LSRFortessa cell analyzer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software v.10.7.1 (FlowJo, Ashland, OR, USA).

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.