Fitc conjugated anti cd56

Co-cultured NK cells were stained using the following four monoclonal antibodies (mAb): APC- or PE-conjugated anti-CD3, PE-cy7-conjugated anti-CD16, FITC-conjugated anti-CD56, and BV510-conjugated anti-PD-1 mAbs (BD Biosciences). NK cell analyses were performed using a BD FACSVerse flow cytometer (BD Biosciences) equipped with three lasers (488 nm, 630 nm, and 405 nm). Data were analyzed using the FlowJo 10 Software (TreeStar Inc., USA).

FITC-conjugated major histocompatibility complex (MHC) class I–specific antibody (IgG2a, W6/32, CBL139F, Cymbus Biotech, Hants, UK) and PE anti-human CD155/PVR (clone SKIL.4, Biolegend, Cat No 337609) were used for flow cytometry. FACSAria II Cell Sorter 8-colour flow cytometer with BD FACSDiva™ 8.0 Software (BD Biosciences, San Jose, CA) was daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; BD Biosciences). PerCP-Cy5.5 anti-CD3 (BD Biosciences Cat No 560835), FITC-conjugated anti-CD56 (BD Biosciences, Cat No 345811) and PE-conjugate anti-CD107a antibody (BD Bioscience, Cat No 555801) were used to identify degranulating NK-cells from peripheral blood. For intracellular staining PE-conjugated anti-IFN-γ antibody (BD Biosciences Cat No 554701) was used. Fluorochrome-labelled monoclonal antibodies for identification of tissue Treg cells were: FITC-anti-FOXP3 (BD Biosciences, Cat No 560047), Pe-Cy7-anti-CD25 (BD Biosciences, Cat No 557741) and APC-Cy7-anti-CD4 (BD Biosciences, Cat No 557871). Intracellular staining for FoxP3 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.