Streptavidin bv510

PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described (Juno et al., 2020 (link)). Probes were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher Scientific) to trimeric S protein biotinylated using recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labeled to APC using an APC Conjugation Lightning-Link Kit (Abcam). PBMCs were surface stained with Aqua viability dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U, Beckman Coulter), IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048), CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829) (BioLegend). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII Fortessa.

Probes for delineating SARS-CoV-2 S-specific B cells within cryopreserved human PBMCs were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher) to trimeric S protein biotinylated using recombinant Bir-A (Avidity). Cells were stained with Aqua viability dye (Thermo Fisher) in PBS. Cells were then stained with S-PE probes and surface monoclonal antibodies in 1% FCS in PBS for 30 mins at 4°C. Monoclonal antibodies for surface staining included CD19-ECD (J3-119, 1:150) (Beckman Coulter), IgM BUV395 (G20-127, 1:150), CD21 BUV737 (B-ly4, 1:150), IgG BV786 (G18-145, 1:75), streptavidin-BV510 (1:600), CD11c (B-ly6, 1:100) (BD Biosciences), CD20 APC-Cy7 (2H7, 1:150), CD14 BV510 (M5E2, 1:300), CD3 BV510 (OKT3, 1:600), CD8a BV510 (RPA-T8, 1:1500), CD16 BV510 (3G8, 1:500), CD10 BV510 (HI10a, 1:750) and CD27 BV605 (O323, 1:150), CD71 PeCy7 (CY1G4, 1:100) (BioLegend), IgD AF488 (Goat polyclonal, 1:100) (Southern Biotech), IgA VioBlue (IS11-8E10, 1:100) (Miltenyi Biotec). Cells were washed, fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSR Fortessa.