Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell‐mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin‐granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)‐α pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF‐α act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell‐mediated cytotoxicity induced by CTLs or NK cells is described.

The fluorochromes used are: PKH‐26, a stable membrane dye for the labeling of the effector cells, annexin V‐FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells.

By eliminating through gating PKH‐26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL‐2 stimulated PBL against K562, Jurkat and KYM‐1 was evaluated.

This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell‐mediated cytotoxicity. Cytometry 37: 197–204, 1999. © 1999 Wiley‐Liss, Inc.