Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species

Background: The most common assay used to detect natural killer (NK) and cy totoxic T-lymphocyte (CTL) activity is the Cr-51 release assay. The numerou s disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. Methods: This assay is based on a dual fluorescent staining of target cells . The dye, DIOC18((3)) (3,3'-dioctadecyloxacarbocyanine perchlorate), is us ed to stain the membrane of different target cells. Propidium iodide (PI) i s used to label dead target and effector cells. This labeling allows a clea r discrimination between both cell populations. Results: A good correlation was observed between the percentage of target l ysis and the effector-to-target cell (E/T) ratios with human and porcine pe ripheral blood mononuclear cells (PBMC) as effector cells. The flow cytomet ric assay was shown to be as sensitive and as reliable as the Cr-51 release performed with human cells. The assay was also applied successfully to mea sure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. Conclusions: We provide evidence that the now cytometric assay using DIOC18 ((3)), is highly reproducible and is suitable to measure different types of cell cytotoxicity. Cytometry 41:289-297, 2000. (C) 2000 Wiley-Liss, Inc.