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Background The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the 51Cr release assay. The numerous 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 used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. Results A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the 51Cr release performed with human cells. The assay was also applied successfully to measure 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 flow cytometric assay using DIOC18(3) is highly reproducible and is suitable to measure different types of cell cytotoxicity. Cytometry 41:289–297, 2000. © 2000 Wiley-Liss, Inc.

The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous 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.This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations.A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus.We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.