NK Cell Cytotoxicity and Cytokine Profiling

NK cell responses to target cell lines were determined as previously described [13] (link). Briefly, mononuclear cells were incubated with MHC class I-devoid K562, 721.221, or antibody-coated p815 cell lines (ATCC) at an effector to target ratio of 10∶1. CD107a-PECy5 (10 µl/ml), brefeldin A (0.5 µg/ml, Sigma-Aldrich) and monensin (0.5 µg/ml, GolgiStop; BD Biosciences) were added at the start of the incubation. After five hours, cells were washed and stained to identify NK cells as above. Cells were fixed, permeabilized and stained for intracellular cytokines using MIP1β-PE and IFNγ-FITC (both BD Biosciences) antibodies. Multi-parameter flow cytometry was performed on a 4 laser BD LSRII system and analyzed as above.