NK Cell Cytotoxicity Assay Protocol

NK-cell responses to target cell lines were determined as previously described [18 (link)]. PBMC were incubated with culture media alone or with K562 cells, 221 cells or antibody-coated p815 cells at an effector to target ratio of 10:1 for 5 hours in the presence of anti-CD107a PE-Cy5 (BD Biosciences, 10 ml/mL), brefeldin A (2.5 mg/mL) and Golgistop (2.5 mg/mL, BD Bioscience). Cells were then washed, stained with the Fixable Blue Dead Cell Stain prior to staining for surface receptors, then fixed, permeabilized (BD Fix/perm, BD Biosciences) and stained with anti-IFNγ-FITC, anti-MIP1β-PE and anti-TNF-Alexafluor700 (all BD Biosciences). Gates were set on media-only controls, which were also used for background subtraction prior to analysis.