So lsrfortessa x20 flow cytometer

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).

Two million of PBMCs were incubated with BD human FC block (BD Biosciences) and then stained with recombinant biotinylated spike S1 + S2 ECD-His (Sino Biological) conjugated with SA-R-Phycoerythrin (PE) and recombinant biotinylated-receptor binding domain  (RBD; BioLegend) conjugated with SA-Allophycocyanin (APC), together with the following fluorescent antibodies (clone; dilution used): CD3-BV650 (clone OKT3; 1:200); CD21-FITC (clone B-LY4; 1:50), CD19-BUV395 (clone SJ25C1; 1:100), CD10-PECF594 (clone HI10A; 1:200), IgM-BV605 (clone G20-127; 1:50), IgD-BV711 (clone IA6-2; 1:100), CD27-BV786 (clone O323; 1:50), CD11c-BB700 (clone 3.9; 1:50), CD20-APCH7 (clone 2H7; 1:100), CD38-BUV737 (clone HB7; 1:400), IgG-PE-Cy7 (clone G18-145; 1:100; all from Becton Dickinson), IgA-Vio blue (clone IS11-8E10; 1:100; Miltenyi Biotec). Following surface staining, cells were washed once with PBS and labeled with Zombie Aqua Fixable Viability Kit (Thermofisher) according to the manufacturer instruction. Cells were fixed in BD fixation solution (BD Biosciences) and acquired with SO LSRFortessa X20 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v10 (TreeStar, USA).

A 7-color panel was developed to phenotype B-cell populations and identify SARS-CoV-2-specific B cells among PBMC by flow cytometry. The biotinylated spike proteins were tetramerized with fluorescently labeled streptavidin (SA) as follows: Spike S1+S2 ECD-His recombinant biotinylated-protein (Sino Biological) with SA-R-Phycoerythrin (PE), RBD recombinant biotinylated-protein (BioLegend) with SA-Allophycocyanin (APC). Two million PBMCs were incubated with BD human FC block (BD Biosciences) for 10 min at RT, and stained for 30 min at 4°C with the following antibody-fluorochrome panel: CD3-BV650 (clone SK7); CD19-BUV395 (clone SJ25C1), IgD-BV711 (clone IA6-2), CD20-APCH7 (clone 2H7), CD27-BV786 (clone M-T271), CD38-BUV737 (clone HB7, all from Becton Dickinson). Following surface staining, cells were washed once with PBS and labeled with Live/Dead Zombie according to the manufacturer instruction (Thermofisher). Cells were washed once with PBS, resuspended in 100 µL BD fixation solution (BD Biosciences) and incubated at 4°C for 15 min in the dark. All antibodies were titrated for optimal dilution. About 1-2 × 10⁶ cells were acquired and stored for each sample with SO LSRFortessa X20 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v10 (TreeStar, USA).