title: Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing.
abstract: We have developed a highly sensitive biophotonic luciferase assay as an alternative to (51)Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene (fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo(+) target cells with CD8(+) CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by (51)Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.
authors.affiliation: Division of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, CA 91010, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Brown
authors.forename: Christine L
authors.initials: CL
authors.lastname: Wright
authors.forename: Araceli
authors.initials: A
authors.lastname: Naranjo
authors.forename: Reena P
authors.initials: RP
authors.lastname: Vishwanath
authors.forename: Wen-Chung
authors.initials: WC
authors.lastname: Chang
authors.forename: Simon
authors.initials: S
authors.lastname: Olivares
authors.forename: Jamie R
authors.initials: JR
authors.lastname: Wagner
authors.forename: Laura
authors.initials: L
authors.lastname: Bruins
authors.forename: Andrew
authors.initials: A
authors.lastname: Raubitschek
authors.forename: Laurence J N
authors.initials: LJ
authors.lastname: Cooper
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jensen
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D014162:Transfection
chemical_list: D049409:Luciferases, Firefly