Anti 2b4 fitc

The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).

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.).

Flow cytometry of NK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Staining of NK cells was performed with four panels, and the antibodies used were: anti-CD56-BV605, anti-NKG2D-APC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-4-1BB-PerCP-Cy5.5, anti-CD95-BV421, anti-Tim3-BV421, anti-TRAIL-PE, anti-CD122-PerCP-Cy5.5, anti-CD122-BV510, anti-CD95L-PE, anti-Ki67-BV421, anti-CD40L-BV510, anti-PD-1-PE-Cy7; all were obtained from BioLegend (San Diego, CA). Anti-CD16-APC-H7, PD-L1-PE-Cy7, and CD11a-FITC, were obtained from BD Biosciences (San Jose, CA). Anti-NKG2A-PE was from R&D Systems (Minneapolis, MN), and anti-CD158a-PerCP-Cy5.5 from eBioscience.