Streptavidin apc

Recombinant SARS-CoV-2 S-2P trimer was biotinylated using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (ThermoFisher) and tetramerized with streptavidin-APC (Agilent) as previously described (Krishnamurty et al., 2016 (link); Taylor et al., 2012 (link)). The RBD domain of SARS-CoV-2 S was biotinylated and tetramerized with streptavidin-APC (Agilent). The APC decoy reagent was generated by conjugating SA-APC to Dylight 755 using a DyLight 755 antibody labeling kit (ThermoFisher), washing and removing unbound DyLight 755, and incubating with excess of an irrelevant biotinylated His-tagged protein. The PE decoy was generated in the same manner, by conjugating SA-PE to Alexa Fluor 647 with an AF647 antibody labeling kit (ThermoFisher).

The following antibodies or staining reagents were purchased from BioLegend: CD19 (SJ25C, 363028), CD3 (OKT3, 317328), CD11C (3.9, 301608), CD25 (M-A251, 356126), CD14 (M5E2,301836), and IgM (IgG1-k, 314524). CCR7 (150503, 561143) and IgG (G18-145, 561297) were obtained from BD Bioscience, CD45RA (T6D11, 130-113-359) from Miltyeni Biotech, and CD8A (SK1, 48-0087-42) from eBiosciences. The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit was obtained from Invitrogen. Biotinylated spike protein expressed and purified as previously described^{26 (link)} was conjugated to streptavidin R-phycoerythrin (PJRS25-1) or streptavidin APC obtained from Agilent Technologies. PBMCs were isolated from study participants and stored in liquid nitrogen. Aliquots containing 10⁷ cells were thawed and stained in PBS containing 2 mM EDTA at 4 °C with the above antibody panel and then transferred to 0.04% BSA in PBS. Events were acquired on a FACSAria Fusion (BD Biosciences). Analyses were carried out in FlowJo version 10.7.1.