Nkp30

CellCarrier Ultra tissue culture treated plates (Perkin Elmer) were coated with either 0.01% PLL (Merck) or a combination of 2 µg/ml ICAM-1 (R&D Systems), 1 µg/ml NKp30 (R&D systems, MAB18491) and 1 µg/ml NKp46 (BD Biosciences, 557487). NK-92 cells were cultured in IL-2 free medium overnight. 15000 NK-92 and 5000 primary NK cells were seeded per well and left for 30 min at 37°C to adhere and form the synapse. Cells were fixed with 3% paraformaldehyde (Merck) and stained with anti-perforin Ab and phalloidin-AF 488. CellCarrier Ultra multiwell tissue culture treated plates were coated with either 0.01% poly-L-lysine or a combination of 2 µg/ml ICAM-1 and 10 µg/ml anti-CD3 (eBioscience). 10000 Jurkat or 5000 CD8 + T cells were seeded per well and left for 15 min at 37°C to adhere and form the synapse. Cells were fixed with 3% paraformaldehyde and stained with anti-LFA-1 (BioLegend, 301202) and phalloidin-AF 488 (Thermo Fisher Scientific) in permeabilization buffer (eBioscience). Goat anti-mouse AF 647 antibody (Thermo Fisher Scientific, A-21240) was used to reveal LFA-1 staining. CD8 + T cells were in addition stained with anti-perforin and goat anti-mouse AF 555 (Life technologies) was used to reveal perforin staining. Nuclei were stained with DAPI (Thermo Fisher Scientific). Stained cells were kept in PBS at 4°C until imaging.