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 inher ent problems that include hazards associated with radioactivity, cell label ing and high spontaneous release. Here we describe a novel how cytometric a ssay which addresses and improves upon the problems currently encountered w ith the Cr-51 release assay. The fluorometric assessment of T lymphocyte an tigen specific lysis (FATAL) assay employs dual staining (PKH-26 and CFSE) to identify and evaluate the target population. We found that the PKH-26/CF SE combination efficiently labeled target cells. Evaluation of the spontane ous leakage from dye labeled target cells was forty fold lower than the spo ntaneous leakage seen with the Cr-51 release assay. The FATAL assay permitt ed a more accurate assessment or the effector: target ratio, and detected l ow levels of cytotoxic T lymphocyte (CTL) mediated lysis. There was a stron g correlation between the Cr-51 release and FATAL assays, when performed in parallel with identical effector and target cells (r(2) = 0.998, P = <0.00 01). This novel method of detecting cytolysis represents a qualitative and quantitative improvement over standard Cr-51 release analysis. The FATAL as say will be of value to further investigate mechanisms of cytolysis by effe ctor cell populations. (C) 2001 Elsevier Science B.V. All rights reserved.