Automated fluorometric assay for T cell cytotoxicity.

A fluorometric assay avoiding the use of radioactivity has been developed for detecting cytotoxic T lymphocytes (Tc cells). The method involves labelling targets with Hoechst dye no. 33342 (H33342) which becomes brightly fluorescent on binding to DNA. Lysis of target cells by Tc cells is quantified by measuring the release of fluorescent H33342 into the supernatant of culture wells. The fluorescence is measured using an automated Microfluor reader which allows results to be obtained rapidly. The assay has been used to detect alloreactive Tc cells and H-2 restricted Tc cells against influenza virus in a short-term 6 h assay using P815 and L929 as targets with comparable results to those obtained with 51Cr labelling. In contrast, lymphocyte blasts were found to be less sensitive in 6 h fluorometric assays when compared with the 51Cr assay. In long-term overnight assays (possible because of the low spontaneous release of H33342 from targets) lymphocyte blasts gave high specific lysis and some anti-self reactivity. The cause of the anti-self reactivity may reflect fundamental differences between the H33342 and 51Cr release assays.