Flow cytometry

Antibodies were obtained from BioLegend (San Diego, California), Columbia Biosciences (Frederick, Maryland), BD Biosciences (San Jose, California), Miltenyi Biotec (Bergisch Gladbach, Cologne), eBioscience (Carlsbad, California) or ThermoFisher Scientific (Waltham, Massachusetts): CD38-phycoerythrin (PE)-Cy7 (clone HIT2); IgE-allophycocyanin (APC) (Columbia Biosciences SKU: D3–110-E); IgG-PE (clones G18–145 and HP6017, and ThermoFisher Scientific Catalog #12–4998-82); IgA-PE (clone IS11–8E10); IgA-APC (clone IS11–8E10); IgM-Brilliant Violet (BV) 510 (clone MHM-88); IgD-BV421 (clone IA6–2); IgG-biotin (ThermoFisher Scientific Catalog #A18815); CD20-Alexa Fluor700 (clone 2H7); CD27-FITC (clone O323). In all assays 1 × 10⁶ cells were first incubated with Human TruStain FcX (Fc Receptor Blocking Solution, Biolegend) or anti-human CD32 (FcγRII Blocker, StemCell Technologies) for 15 minutes on ice to block non-specific staining and then incubated with fluorochrome-conjugated antibodies for 30 minutes on ice and protected from light. When IgG-biotin was used to label IgG⁺ cells, cells were incubated for an additional 30 minutes with streptavidin-PE (BioLegend) on ice and protected from light. Dead cells were excluded using the fixable viability dye eFluor780 (eBioscience) and by gating on singlets. FMO were used for gating. Data were acquired on a Fortessa (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore), and single cells were sorted on a MoFlo XDP Cell Sorter (Beckman Coulter).