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Flow cytometric cytotoxicity assay for measuring mammalian and avian NK cell activity
Csaba Vizler1, Tünde Nagy, Erzsébet Kusz
1Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary. vizler@nucleus.szbk.u-szeged.hu
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 propidium iodide (PI), and flow-cytometric analysis was performed.
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.
Insights
Flow-cytometry offers a convenient method for natural killer (NK) cell assays. This study demonstrates MitoTracker Green FM and propidium iodide (PI) staining effectively measures NK cell cytotoxicity, even in avian models.
Area of Science:
- Immunology
- Cell Biology
Background:
- Flow-cytometry provides a practical alternative to traditional radioactive natural killer (NK) cell assays.
- MitoTracker Green FM, a mitochondrial stain, shows potential for labeling NK target cells.
- Propidium iodide (PI) is used to identify dead cells.
Purpose of the Study:
- To evaluate MitoTracker Green FM and PI staining for flow-cytometric NK cell assays.
- To assess the efficacy of this method in both murine and avian models.
Main Methods:
- NK target cells were labeled with MitoTracker Green FM.
- Effector spleen cells were incubated with labeled target cells.
- Cells were stained with PI and analyzed via flow cytometry to distinguish live and dead cells.
Main Results:
- MitoTracker Green FM effectively stained all cell types, including resting cells, and was retained by dead cells.
- Double staining with MitoTracker Green FM and PI enabled accurate discrimination of live and dead target cells.
- Cytotoxicity values in mouse models were consistent with expected ranges.
- Chicken fibroblasts exhibited significant sensitivity to autologous splenic NK cells, comparable to classic NK targets.
Conclusions:
- MitoTracker Green FM and PI staining facilitate convenient and reliable flow-cytometric NK cell assays.
- This method confirms chicken fibroblasts are susceptible to autologous NK cell-mediated cytotoxicity.