Cytokine release assay

NALM6 cells were seeded in tissue culture-treated 96-well plates (Cat.: 3879, Corning Incorporated) at a density of 10,000 cells/well. NK cell engagers were added at a final concentration of 10 nM and incubated at 37°C for 30 min. NK cells were then added at E:T ratio of 5:1, and the plates were incubated for 24 h at 37°C and in a 5% CO₂ atmosphere in a humidified incubator. Plates were centrifuged at 1,000×g for 5 min, and cell culture supernatants were harvested for quantification of human IFN-γ (Cat.: DIF50C, R&D Systems) and human TNF (Cat.: DTA00D, R&D Systems) according to the manufacturer’s protocols.