Igd buv395

B cells were incubated with an antibody cocktail containing L/D marker (eBioscience Fixable Viability Dye eFluor 780) and antibodies CD10 BUV737 (BD #612826), CD20 PECy7 (BioLegend #302312) and CD27 BV711 (BioLegend #302834) and IgD BUV395 (BD #563813) for 30 min at 4°C. Labelled B cells were sorted into naïve (CD10⁻CD20⁺CD27⁻IgD⁺) and memory (CD10⁻CD20⁺CD27⁺ and CD10⁻CD20⁺CD27⁻IgD⁻) B cells using the FACSAria™ Fusion sorter (BD Biosciences) (gating strategy shown in Figure S1A).

For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then surface stained with the relevant probes (APC or PE labelled stem, HEL, OVA or gp120 proteins for baiting antigen-specific GC B cells) [10 (link)] and the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend). For detection of Tfh cells ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend). For Ag-specific Tfh identification [23 (link)], cells were cultured as described above and then stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

To test the specificity of our approach, COVID-19⁺ and prepandemic controls were stained with dCODE Dextramer–PE complexes, as well as complexes of SARS-CoV-2 S and N coupled with fluorophore-streptavidin conjugates (APC, BioLegend, catalog 405207). Following addition of anti–human CD19 APC-H7 (BD Biosciences, catalog 115530), IgD BUV395 (BD Biosciences, catalog 563813), and LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, catalog L34966), cells were acquired on BDFACSAria Fusion X20.
For sorting of SARS-CoV-2–specific B cells, PBMCs were stained with TotalSeq anti–human Hashtag Antibody and Human TruStain FcX (BioLegend), similarly to the other 2 fractions. Cells were subsequently stained with dCODE Dextramer–PE complexes, as well as complexes of SARS-CoV-2 S or N coupled with fluorophore-streptavidin conjugates (APC and BV421, BioLegend, catalog 405225) in order to reduce background binding. Following addition of anti–human CD19 APC-H7, IgD BUV395, and LIVE/DEAD Fixable Aqua Dead Cell Stain, cells were sorted using a BDFACSAria Fusion. The whole sample was sorted.