Anti cd8 apc efluro 780

PBMCs from sample 111 were enriched for B cells via positive selection using CD19 microbeads (Miltenyi Biotec; 130–050-301). PBMCs from all other donors were enriched for B cells by negative selection (Miltenyi Biotec; 130–101-638). All selection protocols were performed according to the manufacturer’s instructions. Enriched B cells were incubated for 30 min on ice in FACS buffer (1× PBS, 2% calf serum, 1 mM EDTA) with fluorophore-labeled EDIII and ovalbumin, and in the presence of anti-human antibodies anti-CD3-APC-eFluro 780 (Invitrogen; 47–0037-41), anti-CD8-APC-eFluro 780 (Invitrogen; 47–0086-42), anti-CD14-APC-eFluro 780 (Invitrogen; 47–0149-42), anti-CD16-APC-eFluro 780 (Invitrogen; 47–0168-41), anti-CD20-PECy7 (BD Biosciences; 335793), and Zombie NIR (BioLegend; 423105). Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR⁻CD20⁺Ova⁻EDIII-PE⁺EDIII-AF647⁺ B cells were sorted using a FACS Aria III (Becton Dickinson) into individual wells of 96-well plates. Each well contained 4 µl of a lysis buffer comprising 0.5× PBS, 10 mM DTT, and 3,000 U/ml RNasin Ribonuclease Inhibitors (Promega; N2615). Sorted cells were snap-frozen on dry ice and then stored at −80°C. Antibody sequences are derived from memory B cells because they originate from small CD20⁺ cells, and the antibody genes were PCR amplified using IgG-specific primers.

As previously described (Robbiani et al., 2020 (link)), PBMCs were enriched for B cells via negative selection using a pan–B cell isolation kit (130-101-638; Miltenyi Biotec) according to the manufacturer’s protocol. Enriched B cells were incubated with fluorophore-labeled RBD and ovalbumin, and in the presence of antihuman antibodies anti-CD3-APC-eFluro 780 (47-0037-41; Invitrogen), anti-CD8-APC-eFluro 780 (47-0086-42; Invitrogen), anti-CD14-APC-eFluro 780 (47-0149-42; Invitrogen), anti-CD16-APC-eFluro 780 (47-0168-41; Invitrogen), anti-CD20-PECy7 (335793; BD Biosciences), and Zombie NIR (423105; BioLegend) in FACS buffer (1 × PBS, 2% calf serum, 1mM EDTA) for 30 min on ice. Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR^-CD20⁺Ova⁻RBD⁺RBD KEN⁺ were sorted using a FACS Aria III (Becton Dickinson) into individual wells of a 96-well plate, each containing 4 μl of lysis buffer comprising 0.5× PBS, 10 mM dithiothreitol, and 3,000 U/ml RNasin Ribonuclease Inhibitors (N2615; Promega). Sorted cells were frozen on dry ice and stored at −80°C until further processing.