A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis

The Cr-51 release assay has traditionally been used to investigate effector cell cytotoxic function against labeled targets, but this method has inherent problems that include hazards associated with radioactivity, cell labeling and high spontaneous release. Here we describe a novel how cytometric assay which addresses and improves upon the problems currently encountered with the Cr-51 release assay. The fluorometric assessment of T lymphocyte antigen specific lysis (FATAL) assay employs dual staining (PKH-26 and CFSE) to identify and evaluate the target population. We found that the PKH-26/CFSE combination efficiently labeled target cells. Evaluation of the spontaneous leakage from dye labeled target cells was forty fold lower than the spontaneous leakage seen with the Cr-51 release assay. The FATAL assay permitted a more accurate assessment or the effector: target ratio, and detected low levels of cytotoxic T lymphocyte (CTL) mediated lysis. There was a strong correlation between the Cr-51 release and FATAL assays, when performed in parallel with identical effector and target cells (r(2) = 0.998, P = <0.0001). This novel method of detecting cytolysis represents a qualitative and quantitative improvement over standard Cr-51 release analysis. The FATAL assay will be of value to further investigate mechanisms of cytolysis by effector cell populations. (C) 2001 Elsevier Science B.V. All rights reserved.