title: The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers.
abstract: We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.
authors.affiliation: The Queensland Institute of Medical Research and the Cooperative Research Centre for Vaccine Technology, University of Queensland, Brisbane Qld 4029, Australia. norbertK@qimr.edu.au
authors.forename: Norbert
authors.initials: N
authors.lastname: Kienzle
authors.forename: Stuart
authors.initials: S
authors.lastname: Olver
authors.forename: Kathy
authors.initials: K
authors.lastname: Buttigieg
authors.forename: Anne
authors.initials: A
authors.lastname: Kelso
mesh_terms: D000818:Animals; D002460:Cell Line; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011419:Propidium; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection
chemical_list: D002860:Chromium Radioisotopes; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011419:Propidium