Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity

Background: Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are ki lled by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzpme, the Fas ligand (Fas L), and t he Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apo ptosis. The other effecters, Fas L and TNF-alpha act by an apoptosis mechan ism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described . Methods: The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct eval uation of early apoptotic cells and propidium iodide which distinguishes me mbrane permeabilized and late apoptotic cells. Results: By eliminating through gating PKH-26 positive effector cells, we o btain a direct estimation of the percentage of target cells in the early st ages of apoptosis as well as the percentage of target cells dying after lat e apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 s timulated PBL against K562, Jurkat and KYM-1 was evaluated. Conclusions: This rapid and novel assay permits the dis crimination of the cell death mechanisms occurring during a cytotoxic response and to precisel y evaluate the contribution of apoptosis in the early phases of cell-mediat ed cytotoxicity. Cytometry 37: 197-204, 1999. (C) 1999 Wiley-Liss, Inc.