Assessing Licensing of NK Cells

Natural killer (NK) cells express receptors to detect and kill target cells based on expression of target cell surface molecules. Through a process termed NK cell licensing, only NK cells that express inhibitory receptors (e.g., Ly49 receptors in the mouse) for self-major histocompatibility complex (MHC) class I molecules become functionally competent to be triggered through their activation receptors. To determine the licensing status of particular Ly49⁺ murine NK cell subsets, splenocytes are stimulated with plate-bound anti-NK1.1 monoclonal antibody in the presence of brefeldin A and then assessed for NK cell activation on a single-cell basis using intracellular cytokine interferon-γ staining and flow cytometry.