A VERSATILE FLOW CYTOMETRY-BASED ASSAY FOR THE DETERMINATION OF SHORT-TERM AND LONG-TERM NATURAL-KILLER-CELL ACTIVITY

A flow cytometry based method has been developed to assess natural killer (NK) cell activity in both short-term (4 h) and long-term (18 h) NK assays. Target cells were either labeled with PKH-2, c'FDA or D275. Simultaneously, dead cells were identified by counter-staining with the nuclear dye propidium iodide. Using flow cytometry, only D275 in combination with propidium iodide permits the differentiation of four cell populations: live target cells, dead target cells, live effector cells, and dead effector cells. Even after the extended incubation periods (18 h) necessary for the determination of NK activity in some domestic animals these four populations remain clearly distinguishable. Comparison of results with cells of normal human individuals obtained using this D275/propidium iodide flow cytometry assay with data derived from fluorescence microscopy or an endogenous lactate dehydrogenase release assay shows a strong correlation. Since in long-term NK assays a high proportion of dead effector cells is constantly observed this cell population frequently limits the use of the lactate dehydrogenase release assay but does not interfere with the flow cytometry assay presented here. Using this novel assay, we have demonstrated the suppressive effects of defined glycosaminoglycans on long-term porcine NK activity.