Anti dnam 1 pe

The expanded NK cells were stained with the following monoclonal Abs; anti-CD3- fluorescein isothiocyanate (FITC), anti-CD14-FITC, anti-CD16- phycoerythrin (PE), anti-CD19-PE, anti-DNAM-1-PE, anti-CD56-PE-cyanine (Cy)5, anti-CXCR3-PE, anti-NKp30-PE, anti-NKp44-PE, anti-NKp46-PE (all from BD Biosciences, San Jose, CA, USA), anti-NKG2A-PE, anti-NKG2C-PE, and anti-NKG2D-PE (all from R&D systems, Minneapolis, MN, USA). Stained NK cells were acquired on LSR Fortessa and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). To confirm phenotypic changes in NK cell receptors in co-culture of tumor cells with NK cells, cryopreserved NK cells were thawed and co-cultured with MIA PaCa-2 at an E:T ratio of 1:1. On 1, 2, or 3 days after co-culture, the harvested NK cells were immunostained with anti-CD16-PE, anti-NKp30-PE, anti-NKp44-PE, anti-NKp46-PE, anti-DNAM-1-PE, anti-CXCR3-PE (all from BD Biosciences, San Jose, CA, USA), anti-NKG2D-PE (R&D systems, Minneapolis, MN, USA), anti-CD96-PE, and anti-CD161-PE (all from eBioscience, San Diego, CA, USA) then analyzed by flow cytometry, as described above.

We stained cells with appropriate antibodies, which were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using CellQuest software ver 6.0 (BD Biosciences). We stained GBM cell lines using the following mouse anti-human antibodies: anti-CD112-PE (R2.525) and anti-CD50-PE (TU41)—BD Biosciences; anti-MIC-A-PE (AMO1) and anti-MIC-B-PE (BMO1)—MBL; anti-ULBP-1-PE (170818), anti-ULBP-2-PE (165903), and anti-ULBP-3-PE (166510)—R&D Systems (Minneapolis, MN); and anti-HLA-ABC-PE (W6/32), anti-HLA-E-PE (3D12), anti-CD48-PE (156-4H9), anti-CD155-PE (2H7CD155), anti-CD54-PE (HA58), and anti-CD102-PE (CBRIC2/2)—Thermo Fisher Scientific (Waltham, MA).
The expanded NK cells were stained with the following antibodies: anti-CD3-FITC (UCHT1), anti-CD56-PE-Cy5 (B159), anti-CD11a-PE (HI111), anti-CD16-PE (3G8), anti-DNAM-1-PE (DX11), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), and anti-CD161-PE (NKR-P1A)—BD Biosciences; anti-CD158a-PE (EB6Bf) and anti-CD159a-PE (NKG2A)—Beckman Coulter (Pasadena, CA); anti-NKG2D-PE (149810)—R&D Systems; anti-CD2-PE (RPA-2.10), anti-CD4-PE (OKT4), anti-CD8-PE (SK1), anti-CD244-PE (C1.7), anti-CD94-FITC (DX22), and anti-CD158b-PE (GL183)—Thermo Fisher Scientific.