Cd3 bb700

The staining method used for the detection of RBD and Spike memory B cells has been described previously.³² (link) In brief, biotinylated RBD and Spike proteins were incubated with Streptavidin-PE (SA-PE; Molecular probes; ThermoFisher Scientific) and Streptavidin-APC (SA-APC; BD Pharmingen) in a molar ratio of 4:1 and 2:1, respectively. Cryopreserved PBMCs were thawed rapidly in a 37°C water bath and washed with pre-warmed RPMI media supplemented with 2 mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 10% heat-inactivated fetal calf serum (Sigma) then washed twice. A maximum of 1 × 10⁷ cells were stained with Fixable Viability Stain 700 (FVS700) (BD Bioscience in a 1:1000 dilution), 5 μL Human Fc block (BD Bioscience) per 2 × 10⁶ cells, 1 μg/mL each of RBD and Spike tetramers, 5 μL each of CD21 BV421, IgD BV510, CD10 BV605, CD19 BV711 and CD20 APC-H7, 8 μL of IgG BV786, 2 μL each of CD27 PE-CF594 and CD38 PE-Cy7, 2.5 μL HLA-DR BB515 and 0.5 μL CD3 BB700 (BD Bioscience). Cells were washed, resuspended in FACS wash buffer and the data acquired on BD FACSAria™ III. Data analysis was performed using FlowJo version 10.7.1 (TreeStar). To establish background B cell levels for each of antigens and phenotypes we performed the same analysis with PBMCs from 9 healthy control individuals. Background was calculated as: average value + 2SD.