Facs aria sorp 4 laser

biotinylated SARS-CoV-2 S protein and RBD were conjugated to streptavidin-bound fluorophores. Briefly, the biotinylated proteins were incubated for a minimum of 1 h at 4°C with the streptavidin-conjugates AF647 (Biolegend), BV421 (Biolegend) and BB515 (BD Biosciences) at a 1:2 protein to fluorochrome ratio. The fluorescent probes were incubated for at least 10 min with 10mM biotin (GeneCopoeia) to saturate the unconjugated streptavidin-fluorochrome complexes. Cryopreserved macaque PBMCs from 2 weeks after final immunization (week 12) were thawed and counted. 5x10⁶ cells were stained for 30 min at 4°C with the fluorescent probes, a viability marker (LiveDead-eF780, eBiosciences) and the following B cell-specific antibodies: anti-CD20-PE-CF594 (clone 2H7; BD Biosciences), anti-IgG-PE-Cy7 (clone G18-145;BD Biosciences), anti-CD27-PE (clone M-T271; BD Biosciences), and anti-IgM-BV605 (clone MHM-88; Biolegend). Cells were washed twice with FACS buffer and acquired on the FACS-ARIA-SORP 4 laser (BD Biosciences). Analysis was performed on FlowJo v.10.7.1.

To generate probes for B cell sorting, biotinylated GPC-I53-50A from lineage II and IV as well as a biotinylated HCV E1E2-I53-50A (K.S. et al., unpublished data) were conjugated to the streptavidin-bound fluorophores AF647, BV421, and BB515, respectively. Conjugation was performed by incubating the biotinylated proteins for a minimum of 1 h at 4°C with the streptavidin-conjugates at a 1:2 protein to fluorochrome ratio. To saturate unconjugated streptavidin-fluorochrome complexes the fluorescent probes were next incubated for at least 10 min with 10mM biotin (GeneCopoeia) to saturate the unconjugated streptavidin-fluorochrome complexes. Week 29 rabbit PBMCs were then counted and 5x10⁶ cells were stained for 30 min at 4 with the fluorescent probes, a viability marker (LiveDead-eF780, eBiosciences), and a rabbit PE-conjugated anti-IgG marker (Biolegend). Prior to their acquisition on the FACS-ARIA-SORP 4 laser (BD-Biosciences), cells were washed twice with FACS buffer. Viable IgG+ B cells that were negative for HCV E1E2-I53-50A and showed dual staining for both GPC-I53-50A from lineage II and IV were sorted. Analysis was performed on FlowJo v.10.7.1.