Evaluating NK Cell-Mediated Cytotoxicity

Cryopreserved CBMC and PBMC samples were thawed and rested overnight (18 h) at 37°C 5% CO₂ in RPMI 1640 media supplemented with 10% FBS, or media supplemented with 10 ng/mL recombinant human IL-15 (Miltenyi Biotec), as previously described (32 (link)). Subsequently, NK cells were purified by negative selection with magnetic beads (human NK cell isolation kit, Miltenyi Biotec) and used as effector cells (NK cell to target cell ratio of 5:1) in redirected cytotoxicity assays using HIV × CD16 DART molecules, performed as described above. Positive responses were defined as specific killing >20%, and data were not fit to a dose-response function for graphing due to the presence of a prozone (37 (link)).