Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells. This process is accomplished by various immune effector mechanisms, including natural killer leukocytes (NK). NK activity is facilitated by non-specific lysis of NK-receptor-infected targets, or the FcγII receptor (CD16), recognizing IgG bound to specific antigens on the target cell surface. NK cells can also induce apoptosis in target cells. The activity of natural killer cells and their effect on target cells are frequently studied in immunomodulation experiments.
Flow cytometry tests have been developed to overcome some of the difficulties associated with older assays such as lactate dehydrogenase and 51 Cr release assays.
CFSE, a green fluorescent membrane dye, is used to impart a green fluorescence characteristic to the target cell population. The unstained effector cells are then added and incubated with the target cells
At the end of the incubation, the second reagent, 7-aminoactinomycin D (7-AAD), fluorescent red dye, is added to stain all the necrotic cells by binding to the DNA of cells with membrane degeneration.
Since all target cells are initially labeled with CFSE fluorescent green and the effector cells are not, these two populations can easily be distinguished. Control populations are used to compensate for the flow cytometer. Proper synchronization of the flow cytometer easily distinguishes living and necrotic cells in a single sample tube.
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