Facscanto 2 triple laser flow cytometer

PBL subsets were determined as described previously [2 (link), 5 (link)]. For analysis of determinants on the cell surface, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD3, CD4, CD25, CD28, CD62L, CD95, CD119, CD127, CD152, CD154, CD178, CD252, HLA-DR, and CD183 (CXCR3) (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3, IFNγ (clone B27), IL4, IL10, Granzyme B, Perforin, T-bet (all from BD Biosciences), Helios (ebioscience, Frankfurt, Germany) and TGFß₁ (R&D systems, Wiesbaden). 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) [2 (link), 5 (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).

PBL subsets were determined as described previously [24 (link), 25 (link)]. For analysis of cell surface determinants, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD4 (clone RPA-T4), CD25 (clone M - A251), CD127 (clone HIL-7R-M21) (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3 (clone 236A/E7), IFNg (clone B27), CD152 (BN13) and Helios (clone 22 F6) (all BD Biosciences). Briefly, PBL were incubated with combinations of monoclonal antibodies for 30 min and eight-color fluorescence was analyzed using a FACSCanto II triple-laser flow cytometer (BD Biosciences) [24 (link), 25 (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.

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).