Live dead fixable aqua dead cell

To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. 2 × 10⁶ frozen PBMC from SARS-CoV-2 naïve donors were prepared in Falcon® 5ml-round bottom polystyrene tubes at a final concentration of 4 × 10⁶ cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (Seradigm), Penicillin- Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2h at 37°C and 5% CO₂, cells were stained using LIVE/DEAD Fixable Aqua dead cell (Thermo Fisher Scientific, Waltham, MA) in DPBS (GIBCO) at 4°C for 20min. The detection of SARS-CoV-2-antigen specific B cells was done by adding the RBD probes to the antibody cocktail listed in Table S1. Staining was performed at 4°C for 30min and cells were fixed using 2% paraformaldehyde at 4°C for 15min. Stained PBMC samples were acquired on Symphony cytometer (BD Biosciences) and analyzed using FlowJo v10.8.0 software. To avoid excessive noise in phenotyping analyses, we only included donors in whom we detected ≥5 RBD-specific B cells at every time point.