The purity of isolated NK cells (>95%) and expression of surface receptors were determined by flow cytometry using a FACSCalibur (BD-Biosciences). NK cell population was defined as NKp46+CD3− cells.
Changes in the expression of NK cell receptors were examined using the following antibodies: anti-NKp30 PE (Biolegend), anti-NKp44 PE (Biolegend), anti-NKG2D PE (BD-Biosciences), anti-CD16 FITC (Miltenyi Biotec), anti-CD56 FITC and APC (BD-Biosciences), anti-2B4 FITC (Biolegend), anti-NTB-A PE (Biolegend), anti-Dectin-1 PE, anti-TLR-2 PE and anti-TLR-4 PE (BD-Biosciences). Apoptotic NK cells were assessed after an incubation time of 9 h using Annexin V FITC (BD-Biosciences) after staining cells with the surface marker antibodies anti-NKp46 PE, anti-CD3 PerCP and anti-CD56 APC. Intracellular expression of CD56 after incubation with germ tubes for 6 h was investigated by firstly staining CD56 on the cell surface with anti-CD56 FITC (BD-Biosciences). Then, cells were fixed (4% formaldehyde), permeabilized (Wash Perm, BD-Biosciences) and intracellular CD56 was stained using anti-CD56 APC (BD-Biosciences). To remove surface markers, NK cells were incubated in 0.5% trypsin-EDTA (Sigma) for 30 min at 37 °C before samples were stained. All data were analyzed with FlowJo software (Tree Star Inc.).
Changes in the expression of NK cell receptors were examined using the following antibodies: anti-NKp30 PE (Biolegend), anti-NKp44 PE (Biolegend), anti-NKG2D PE (BD-Biosciences), anti-CD16 FITC (Miltenyi Biotec), anti-CD56 FITC and APC (BD-Biosciences), anti-2B4 FITC (Biolegend), anti-NTB-A PE (Biolegend), anti-Dectin-1 PE, anti-TLR-2 PE and anti-TLR-4 PE (BD-Biosciences). Apoptotic NK cells were assessed after an incubation time of 9 h using Annexin V FITC (BD-Biosciences) after staining cells with the surface marker antibodies anti-NKp46 PE, anti-CD3 PerCP and anti-CD56 APC. Intracellular expression of CD56 after incubation with germ tubes for 6 h was investigated by firstly staining CD56 on the cell surface with anti-CD56 FITC (BD-Biosciences). Then, cells were fixed (4% formaldehyde), permeabilized (Wash Perm, BD-Biosciences) and intracellular CD56 was stained using anti-CD56 APC (BD-Biosciences). To remove surface markers, NK cells were incubated in 0.5% trypsin-EDTA (Sigma) for 30 min at 37 °C before samples were stained. All data were analyzed with FlowJo software (Tree Star Inc.).