Cd3 pecy 7 clone sk7

To analyze B cell populations and to identify SARS-CoV-2-specific B cells within PBMC by flow cytometry, a multi-color panel was developed. PBMCs were incubated with the BD human Fc block (BD Biosciences, Aalst, Belgium) for 10 min at RT. The cells were then stained with the SARS-CoV-2 spike full protein ECD-His recombinant biotinylated-protein (25 µg/mL, Sino Biological) in a staining buffer [PBS, 0.5% bovine serum albumin (BSA) and 2 mM EDTA, all from Sigma-Aldrich, St. Louis, MO, USA/Burlington, MA, USA] for 30 min at 4 °C, followed by staining with FITC- and Brillant-Violet-421-conjugated streptavidin for an additional 30 min at 4 °C. Subsequently, the cells were stained for 30 min at 4 °C using the subsequent antibody mixture, comprising CD3-PECy 7 (clone SK7), CD56-PECy7 (clone B159), CD14-PECy7 (clone M5E2), CD19-BUV395 (clone SJ25C1), IgM-BV605 (clone G20-127) and IgD-PE (clone IA6-2) (all from BD Biosciences, Belgium). The cells were then labeled with live/dead FSV780 following the manufacturer’s instructions (BD Biosciences). Finally, the cells were fixed using a BD fixation solution (BD Biosciences) and analyzed with an SO LSRFortessa X20 flow cytometer (BD Biosciences). Data analysis was conducted using FlowJo v10 (TreeStar, Ashland, OR, USA).

Heparinised peripheral blood samples were obtained for analysis of lymphocyte populations and subpopulations by flow cytometry. Whole blood was collected in sodium heparin vacutainers (Becton Dickinson) and shipped ambient overnight to the ITN Flow Cytometry Core (Roswell Park Cancer Institute). Using a stain-lyse method, cells from blinded samples were labelled with 5-colour monoclonal antibody panels using anti-human CD8⁻PE-Cy5, CD57-FITC (clone NK-1), CD56-PE (clone NCA-1) CD14-APC (clone MϕP9), plus CD3-PE-Cy7 (clone SK7, all BD Biosciences). Following staining, data were acquired on a FACSCanto flow cytometer (BD Biosciences), and analysed using WinList’s™ (http://www.vsh.com) FCOM function^{40 (link)}.
A validated panel of KIR receptors^{22 (link)} was used to quantify NK cell subset expression of activating and inhibitory receptors for which clone ids and reagent sources are detailed in Supplementary Data 4. Flow analysis was undertaken using an LSR Fortessa (BD) in the NIHR Cambridge BRC flow phenotyping hub (Supplementary Fig. 9).