Igm percp cy5.5 g20 127

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).

Cryopreserved PBMCs from blood collected 1 and 2 weeks after the H2-F boost were stained with anti-human monoclonal antibodies CD3 BV510 (OKT3, 1:400 dilution, BioLegend, RRID:AB_2561376), CD56 BV510 (HCD56, 1:200 dilution, BioLegend, RRID:AB_2561385), CD14 BV510 (M5E2, 1:200 dilution, BioLegend, RRID:AB_2561379), CD27 BV605 (O323, 1:50, BioLegend, RRID:AB_11204431), CD20 APC-Cy7 (2H7, 1:400 dilution, BioLegend, RRID:AB_314261), IgG BV421 (G18-145, 1:50 dilution, BD Biosciences, RRID:AB_2737665), IgM PercpCy55 (G20-127, 1:40 dilution, BD Biosciences, RRID:AB_10611998), CD19 ECD (H3-119, 1:50 dilution, BD Biosciences, RRID:AB_130854), CD21 PeCy5 (B-ly4, 1:100 dilution, BD Biosciences, RRID:AB_394028) and CD38 (HIT2, 1:400 dilution, BD Biosciences, RRID:AB_1727472). H2 A/Singapore/2/1957 ectodomain and stabilized stem HA probes were expressed, biotinylated and labeled with fluorochromes as described previously (Whittle et al., 2014). Aqua dead cell stain was added for live/dead discrimination (ThermoFisher Scientific). Stained samples were run on a FACS Aria II (BD Biosciences) and data analyzed using FlowJo (TreeStar). CD3- CD14- CD56- CD19+ CD20- CD21- CD27^hi CD38^hi plasmablasts or CD3- CD14- CD56- CD19+ CD20+ IgG+ IgM- Memory B cells were gated, and H2 HA-binding B cells were single-cell sorted into 96-well plates. H2 HA head-specific B cells were identified by indexing.