Flow cytometric cytotoxicity assay for measuring mammalian and avian NK cell activity

Background: Flow-cytometric assays are convenient alternatives to classic radioactive natural killer (NK) tests. MitoTracker Green FM, a green fluorescent intracellular probe serving originally for staining mitochondria, seemed especially suitable for labeling NK target cells. Materials and Methods: NK target cells were labeled with MitoTracker Green FM. After incubation with effector spleen cells, cell suspensions were stained with porpidium was iodide (PI), and flow-cytometric anal formed. Results: MitoTracker Green FM stained efficiently each cell type we assayed, including resting cells, and it was not released from dead cells. NK assays were set up using mouse spleen effector cells and K562 NK target cells. MitoTracker Green FM and PI double staining allowed a discrimination of live and dead target cells, and the cytotoxicity values were in the expected range. Then the method was applied to a less well-known chicken model. We found that chicken-skin fibroblasts had a definite sensitivity to autologous splenic NK cells, sometimes as high as the sensitivity of classic NK targets. Conclusions: Convenient flow-cytometric NK tests can be performed by MitoTracker Green FM and PI staining. Using this method, we demonstrated that chicken fibroblasts are sensitive to the cytotoxic effect of autologous NK cells. (C) 2002 Wiley-Liss, Inc.