Anti nkg2d apc

For CD8⁺NKG2D⁺ cells analysis, cells were harvested and stained with anti-CD3-FITC (E10472-1633, eBioscience, USA), anti-CD8-PE (561946, BD, USA), anti-NKG2D-APC (130-099-216, Miltenyi Biotec, Germany), anti-CD27-PE (560985, BD, USA), and anti-CD57-APC (560845, BD, USA) antibodies. To detect the infiltration of CD8 or NK cells in the tumor mass, tumors were removed and crushed into single cell suspension. Then cells were stained with mouse anti-CD8-PErCp (46-0083-80, eBioscience, USA) or anti-NK1.1-FITC (553164, BD, USA) antibodies. To confirm the depletion of CD8 or NK cells, cells were isolated from peripheral blood and stained with anti-CD8-PErCp (46-0083-80) or anti-NK1.1-PerCp antibodies (45-5941-82) (all from eBioscience, USA). The staining was performed on ice for 30 min. Samples were acquired on the FACS Calibur (BD, USA) and data was analyzed using FlowJo software (Version 8.5.3, Tree Star Inc., USA).

Anti-NKG2D-APC (#130-117-718) and anti-DNAM-1-APC (#130-124-241) were purchased from Miltenyi Biotec (Auburn, CA, USA) and anti-CD16-Pacific Blue (#562874) was purchased from BD Bioscience (San Jose, CA, USA). Cells were stained with antibodies for 20 min at 4 °C. After 20 min of staining, unincorporated dye was removed by washing with 1% FBS containing PBS. Samples were then centrifuged at 1000 rpm for 3 min and the pellets were resuspended in 500 μL of 1% FBS containing PBS. Measurements were performed on a Novocyte flow cytometer (Novocyte, Agilent Technologies, Santa Clara, CA, USA).