Flow Cytometric Characterization of PBL

PBL subsets were determined as described previously and the exact gate setting inclusively FACS plots were described in a recent publication [13 (link)]. For analysis of determinants on the cell surface, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD45, CD3, CD4, CD8, CD16, CD56, CD19, CD25 and CD127 (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3, IFNγ (clone B27) and Helios (ebioscience, Frankfurt, Germany). Briefly, PBL were incubated with combinations of monoclonal antibodies for 30 min as described and eight-color fluorescence was analyzed using a FACSCanto II triple-laser flow cytometer (BD Biosciences) [13 (link)]. When, in addition, intracellular proteins were studied, cell membranes were permeabilized using BD Perm/Wash buffer (BD Biosciences). At least 100,000 events were analyzed in the initial FSC/SSC dot plot. IFNγ monoclonal antibody used for cell separation (BD clone 4S.B3) and IFNγ monoclonal antibody used for cell staining (BD clone B27) were not competitive (data not shown).