Zombie yellow fixable viability staining

For RBD- and S2-specific B cell analysis: Surface staining was performed for 20 min in the presence of 1 mg/mL beriglobin (CSL Behring) with the following fluorochrome-conjugated antibodies titrated to their optimal concentrations: CD20-Viogreen (LT20, Miltenyi), CD14 BV570 (M5E2, Biolegend), CD38 BV605 (HB7, Biolegend), CD27 PE (O323, Biolegend), IgD PerCP-Cy5.5 (IA6-2, Biolegend), CD19 PE-Cy7 (SJ25C1, Biolegend) and CD3 APC-Cy7 (UCHT1, Biolegend) and the labelled proteins (S2 PacBlue (0.25 µg), S2 AlexaFluor488 (0.25 µg), RBD Biotin/Streptavidin PE-Vio615 (0.15 µg) and RBD AlexaFluor647 (0.15 µg)).20 22 (link) Zombie Yellow fixable viability staining (Biolegend) was added during the last 5 min of incubation. After staining, the cells were washed once in PBS/BSA, centrifuged and resuspend in PBS/BSA/2 mM EDTA. For analyses of S-I- and S-II-specific T cells and peripheral blood B and T cell subsets, antibody staining was performed as described before20 22 (link) and in online supplemental methods. All samples were measured on a MACSQuant Analyzer 16 (Miltenyi). Instrument performance was monitored using Rainbow Calibration Particles (BD).