Anti nkp44 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.

DC were membrane-stained with monoclonal anti-IL-15-PE antibody (R&D, Ref: LIT0713051) 2h, 4h, 8h, 24h, 48h and 72h after electroporation of the DC and analyzed on a FACScan flow cytometer (BD). In other experiments, purified NK cells were cocultured at a 5:1 ratio with autologous DC in IMDM + 10% FBS directly after DC electroporation and membrane-stained with anti-CD11c-V450 (BD; Ref: 560369), anti-NKp30-AF360 (BD; Ref: 558408), anti-NKp44-PE (BD; Ref: 558563), anti-NKp46-APC (BD; Ref: 557940), anti-NKG2D-PE (BD; Ref: 557940), anti-CD56-FITC (BD; Ref:345811) and anti-CD69-APC-Cy7 (BD; Ref: 557756) mABs 48h after initiation of cocultures. 7-aminoactinomycin D (7-AAD; BD; Ref: 51-68981E) was used to distinguish between viable and dead cells. Samples were measured on a FACSAria II flow cytometer (BD).