Flow cytometry. (a) Gating strategy for phenotyping. Gating was on lymphocytes singlets that were CD19⁺ or CD20⁺ and CD3⁻CD8⁻CD16⁻Ova⁻. Anti-IgG, IgM, IgA, IgD, CD71, and CD27 antibodies were used for B cell phenotype analysis. Antigen-specific cells were detected based on binding to Wuhan-Hu-1 RBD-PE⁺ and RBD-AF647⁺ or to Wuhan-Hu-1 NTD-BV711⁺ and NTD-BV421⁺. Counting beads were added to each sample and gated based on forward scatter (FSC) and side scatter (SSC) as per manufacturer instructions. (b and c) Representative flow cytometry plots of RBD-binding B cells (b) or NTD-binding B cells (c) in five individuals 1.5 and 6 mo after vaccination. (d and e) Graph showing the frequency of IgM, IgG, and IgA isotype in RBD-specific B cells (d) and NTD-specific B cells (e) 1.5 or 6 mo after vaccination. (f) Gating strategy for single-cell sorting for CD20⁺ B cells for Wuhan-Hu-1 RBD-PE and RBD-AF647 or Wuhan-Hu-1 NTD-BV711 and NTD-BV421. (g and h) Representative flow cytometry plots showing dual Alexa Fluor 647– and PE-Wuhan-Hu-1 RBD binding (g) and BrilliantViolet-711- and BrilliantViolet-421-Wuhan-Hu-1 NTD binding (h); single-cell-sorted B cells from four additional individuals 1.5 or 6 mo after vaccination. Percentage of antigen-specific B cells is indicated.