Igd bv421

Freshly isolated or ex vivo cultured human cells were washed in PBS with 5% bovine serum (FACS buffer) and subsequently stained for 30 minutes at 4°C with fluorophore conjugated antibodies against CD19, CD20, CD22, CD27, or IgD (all BD Biosciences). Cells were then washed twice in FACS buffer and analysed on a FACS Canto2 (BD Biosciences). Where appropriate dead cells were excluded using 4,6-Diamidin-2-phenylindol (DAPI; Thermo Fisher).
To analyse B cells after 9–11 days in culture, plates were spun at 400×g for 3 minutes, supernatants were taken for analysis by ELISA and cells in pellets were re-suspended and pooled for each condition. Cells were washed with PBS and stained with NIR fixable live/dead dye (Molecular probes) for 15 minutes at room temperature and washed with FACS buffer (2%BSA, 2mMEDTA, PBS). Live/dead staining was followed by an incubation with Fc Block reagent (BioLegend) for 10 minutes at RT and finally staining with a mix of labelled antibodies: CD45-BV785, CD19-FITC, CD27-BV711, CD38-APC, CD138-PE, and IgD-BV421 (all BioLegend) following the manufacturers recommendations for 30 minutes at 4C. Stained cells were washed and fixed with 2% PFA in PBS for 10 minutes at RT. Stained cells were analysed in a Beckton Dickinson Fortessa with 355, 405, 488, 561, and 633nm lasers.