| NK effector function has traditionally been examined using 51Chromium release assays. Recently, to avoid costs associated with disposal of radioactive waste, a number of fluorescent based flow cytometric assays have been developed as alternatives. Most of these assays use one dye that is irreversibly incorporated into the cell and propidium iodide as the DNA dead cell staining dye. We describe here a novel assay in which target cells are first labeled or “painted” with CFSE, incubated with effector cells and then monitored for the percentage killed using a membrane impermeant dye, 7AAD. Both live and dead targets retain the signal from the CFSE dye, as it is covalently linked to intracellular amine groups upon internalization and does not leach from cells into the media nor is transferred to adjacent cells. Dead target cells in addition take up the 7AAD. Although propidium iodide has been traditionally used to detect the dead cells, 7AAD is preferable as it better defines the dead cell population, allowing more accurate determination of cell killing. Using this assay, increasing amounts of dead target cells can be detected and quantified as increasing amounts of NK effectors purified from whole blood are added. In addition effects of IL-2 incubation and treatment with anti-perforin antibodies on cell death are readily detectable. |
