Igd pe clone ia6 2

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).

Single-cell suspensions from the BM and peripheral blood were first incubated with human BD Fc block (BD Biosciences) to block Fcγ receptor and then stained with the following anti-human Abs: CD19-APC (clone HIB19, BioLegend), CD34-PE (clone 563, BD Biosciences), CD20-FITC (clone 2H7, BioLegend), CD27-APC/Cyanine7 (clone O323, BioLegend), IgD-PE (clone IA6-2, BD Biosciences), IgM-FITC (clone G20-127, BD Biosciences), CD43-PE (clone CD43-10G7, BioLegend), CD38-PE/Cy7 (clone HIT2, BioLegend), CD38-Percp/Cy5.5 (clone HIT2, BD Biosciences), CD24-PE (clone ML5, BD Biosciences), CD27-V450 (clone M-T271, BD Biosciences), IgD-BV510 (clone IA6-2, BD Biosciences), IgG-PE/Cy7 (clone G18-145, BD Biosciences), Ki-67–PE (clone Ki-67, BioLegend), CD80-FITC (clone L307.4, BD Biosciences), CD86-PE (clone IT2.2, BD Biosciences), HLADR-PE (clone G46-6, BD Biosciences) and CD69-PE/Cy7 (clone FN50, BD Biosciences). 7-AAD Viability Staining Solution (eBioscience, Thermo Fisher Scientific) was used for live versus dead cell discrimination. Samples were analyzed with a FACSVerse flow cytometer (BD Biosciences) using the FACSuite software. Data analysis was performed with FlowJo 10 software (Treestar).