Anti nkp46 antibody

The cytolytic activity of NK cells was evaluated by monitoring the surface expression of CD107a on NK cells upon contact with K562 target cells (15 (link)). Freshly isolated PBMCs were incubated with or without IL-2 (100 Unit/ml) at 37°C overnight. The cells (1 × 10⁶) were then mixed with K562 cells at a serial ratio (as indicated in the Results) in round-bottom 96-well plates and incubated with anti-CD107a mAb (BioLegend) and monensin (eBioscience) at 37°C with 5% CO₂ for 4 h. For evaluation of NK cell degranulation by cross-linking of the receptor NKp46, these cells were incubated in an anti-NKp46 antibody (BioLegend) pre-coated 96-well plates for 4 h. After being washed with PBS–0.2% BSA, cells were stained with anti-CD3, anti-CD16, anti-CD56, and anti-CD57 mAbs. The frequency of CD107a-expressing CD3⁻/CD56⁺CD16⁺ NK cells was analyzed by FCM.

The collected tumors were embedded in OCT (Leica Biosystems, Wetzlar, Germany), frozen in liquid nitrogen, and sectioned into 5-µm slices. Blood vessels were stained with anti-CD31 antibody (Abcam; Cambridge, UK). Macrophages in the tumor sections were stained with anti-F4/80 (Abcam) and anti-CD206 antibodies (BioLegend). Activated NK cells were identified using anti-NKp46 antibody (BioLegend). The obtained confocal images were analyzed with ImageJ 1.48v (NIH, Bethesda, MD, USA), and the results were expressed as the percentage of area (%).