To the editor—The recent article by Liu et al. 1 describes an alternative to 51Cr release for the determination of T-cell–mediated cytotoxicity, using cell-permeable fluorogenic caspase substrates. We agree that measurement of caspase activation in target cells has advantages over measurement of 51Cr release. Caspase-based assays provide mechanistic insight into target cell death and allow evaluation of single cells. However, the assay described by Liu requires flow cytometric analysis immediately after incubation with caspase substrate. This may prove inconvenient after lengthy experiments, or for researchers with limited access to flow cytometry facilities. We have developed a flow cytometric assay for the detection of activated caspase-3 that allows fixation of target cells and delayed analysis. We validated the assay using the human alloreactive cytotoxic T lymphocyte (CTL) clone SKH-13, which recognizes the HLAA* 0201-restricted minor histocompatibility antigen HA-8 (ref. 2). In our assay, target cells are labeled with the red fluorescent probe PKH26 before incubation with CTLs. After incubation, the cells