A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.