Cd20 efluor 450

Dendritic cell-enriched cell suspensions were stained with an antibody cocktail consisting of the following antibodies in PBS + 2% human sera for 30 min on ice: CD1c-APC/Cy7 (L161; BioLegend), CD3-BUV395 (UCHT1; BD Bioscience), CD11b-A700 (M1/70; BioLegend), CD11c-PE/Cy7 (3.9; BioLegend), CD14-A700 (HCD14; BioLegend), CD19-V450 (HIB19; BD Bioscience), CD20-eFluor 450 (2H7; eBioscience), CD56-BV421 (5.1H11; BioLegend), CD123–BV605 (6H6; BioLegend), CD141-BV711 (1A4; BD Bioscience), CD303-PerCP/Cy5.5 (201A; BioLegend), HLA-DR-PE-CF594 (G46-6; BD Bioscience), and NKp46-BV421 (9E2; BioLegend). After washing the cells, they were resuspended in PBS + 2% human sera + 0.1 mg/ml 4′,6-diamidino-2-phenylindole and cell sorted using a BD FACSAria II cell sorter into CD1c⁺ DCs (CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA–DR⁺CD11c⁺CD1c⁺), CD141⁺ DCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11c⁺CD141⁺), pDCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻NKp46⁻HLA-DR⁺CD123⁺CD303⁺), and monocytes (CD3⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11b⁺CD14⁺). The purity of sorted cell populations was reanalyzed and routinely above 95%.

Expression of the following markers on CD4 T cells and CD19 B cells was assessed after 4 days in culture using the following monoclonal antibodies (mAb): CD4-PerCP (Biolegend, San Diego, CA, USA), CD19-APC (BD, Erembodegem-Aalst, Belgium), CD25-PE (DAKO, Glostrup, Denmark), and HLA-DR-FITC (BD). For analysis of B cell differentiation the following monoclonal antibodies were used: CD3-FITC (BD Pharmingen, San Diego, CA, USA), CD19-APC-Alexa Fluor750 (BD), CD20-eFluor450 (eBioscience, San Diego, CA, USA) CD27-APC (ImmunoTools GmbH, Friesoythe, Germany), CD38-PE-CY7 (BD), and IgD-PE (BD). IgG1-FITC/IgG1-PE (Immunotech, Marseille, France) was used as isotype control.
Ki67 intracellular staining was performed using mouse-anti-human Ki67-FITC staining set (eBioscience) following manufactures instructions. Cell acquisition was done using a FACScan flow cytometer and data were analysed with FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).