Brenan M, Parish C R
Department of Immunology, John Curtin School of Medical Research, Canberra, Australia.
J Immunol Methods. 1988 Aug 9;112(1):121-31. doi: 10.1016/0022-1759(88)90042-7.
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.
已开发出一种避免使用放射性的荧光测定法来检测细胞毒性T淋巴细胞（Tc细胞）。该方法包括用Hoechst 33342染料（H33342）标记靶细胞，H33342与DNA结合后会发出明亮的荧光。通过测量荧光H33342释放到培养孔上清液中的量来定量Tc细胞对靶细胞的裂解。使用自动微量荧光读数器测量荧光，从而能够快速获得结果。该测定法已用于在短期6小时测定中检测针对流感病毒的同种反应性Tc细胞和H-2限制性Tc细胞，以P815和L929作为靶细胞，所得结果与用51Cr标记法获得的结果相当。相比之下，发现淋巴细胞母细胞在6小时荧光测定中比在51Cr测定中敏感性更低。在长期过夜测定中（由于靶细胞中H33342的自发释放率低，这是可行的），淋巴细胞母细胞产生了高特异性裂解和一些抗自身反应性。抗自身反应性的原因可能反映了H33342释放测定法和51Cr释放测定法之间的根本差异。
