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Untouched B cells were isolated from human peripheral blood mononuclear cells (PBMCs) using the B Cell Isolation Kit II, human. Isolated B cells were then stimulated with IL-4 and multimeric human CD40L for 48 hours, stained with CD360 (IL-21R) antibodies, and analyzed by flow cytometry using the MACSQuant®
cells are represented in the histograms. The specificity of the conjugated antibodies was confirmed by blocking the binding to the ligand, using pure unconjugated antibodies (left peak). Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies and or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.