Fluorochrome labelled monoclonal antibodies

Flow cytometry was performed on venous peripheral blood collected in heparin-coated vacutainer tubes, on tumor and peritumoral tissues using a FACSCanto II 6-colour flow cytometer, daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; Becton Dickinson, San Jose, CA, USA). Fluorochrome-labelled monoclonal antibodies (BD Bioscience) for identification of circulating and tissue Treg cells were used: Fitc-anti-FOXP3, Pe-Cy7-anti-CD25, APC-Cy7-anti-CD4, APC-anti-CD45RA, Pe-anti-CD152 (CTLA-4), PercP-anti-CD184 (CXCR4), APC-anti-CD279 (PD-1), Pe-anti-CD278 (ICOS) and APC-anti-CD39 (ENTPD1). Intracellular staining for FoxP3, ICOS and CTLA-4 was performed using a commercially available kit (BD Cytofix/Cytoperm; fixation and permeabilization kit; BD Pharmingen) according to the manufacturer's instructions. A minimum of 100.000 events for each sample were collected and data were analysed using FacsDiva software 6.1.3 (BD Bioscience). The absolute number of CD4 was calculated as follow: [total withe blood cell count (cells/uL) x percent CD4]/100 or [total tumor-infiltrating immune cell count (cells/100 mg tumor) x percent CD4]/100.

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).