title: Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.
abstract: We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.
authors.affiliation: Emory Vaccine Center and Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Luzheng
authors.initials: L
authors.lastname: Liu
authors.forename: Ann
authors.initials: A
authors.lastname: Chahroudi
authors.forename: Guido
authors.initials: G
authors.lastname: Silvestri
authors.forename: Mary E
authors.initials: ME
authors.lastname: Wernett
authors.forename: William J
authors.initials: WJ
authors.lastname: Kaiser
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Safrit
authors.forename: Akira
authors.initials: A
authors.lastname: Komoriya
authors.forename: John D
authors.initials: JD
authors.lastname: Altman
authors.forename: Beverly Z
authors.initials: BZ
authors.lastname: Packard
authors.forename: Mark B
authors.initials: MB
authors.lastname: Feinberg
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism*; D002860:Chromium Radioisotopes; D003602:Cytotoxicity, Immunologic / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D002860:Chromium Radioisotopes; D020169:Caspases