Cd56 fluorescein isothiocyanate fitc

Cells were stained for NK cell surface markers with CD56-fluorescein isothiocyanate (FITC) and CD107a-phycoerythrin (PE) antibodies (BD Biosciences) for 30 min at 4°C in 50 μl of staining buffer (PBS + FBS 0.25%). The cells were washed twice with staining buffer. Following surface staining, the cells were resuspended thoroughly; then, 250 μl of fixation/permeabilization solution was added, and the mixture was incubated for 20 min at 4°C. The cells were washed again two times in 1 ml of BD wash buffer. Next, allophycocyanin- (APC-) conjugated anti-TNF-α and anti-IFN-γ antibodies were used to stain the intracellular cytokines of PBMCs for 30 min at 4°C. Corresponding isotype controls were utilized as negative controls. After intracellular staining, the cells were washed twice with 1 ml of BD Perm/Wash buffer and resuspended in staining buffer prior to flow cytometric analysis.

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stored in liquid nitrogen. NK cell purification was performed with the Dynabeads Untouched Human NK cells kit (Life Technologies, Paisley, UK). Antibodies used were: CD56-PECy7, CD3-PerCP, CD16-APC-Cy7, CD57-Pacific Blue (all BioLegend, San Diego, USA), CD56-fluorescein isothiocyanate (FITC) (BD Biosciences, Oxford, UK), CD3-Pacific Blue (eBioscience, Hatfield, UK), CD158a,h-PE, CD158b-FITC, CD158b1/b2,j-PE (all Beckman Coulter, Marseille, France), NKp30-APC, NKp46-APC, NKG2D-APC (all Miltenyi Biotec, Gladbach, Germany), NKG2C-PE, NKG2C-PErCP, NKG2C-APC (all R&D Systems Europe, Oxford, UK), rabbit STAT4 (Invitrogen, Paisley, UK) with goat anti rabbit secondary-APC (Abcam, Cambridge, UK).