title: A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.
abstract: A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.
authors.affiliation: 1 David Braley Human Stem Cell Screening Facility, Stem Cell and Cancer Research Institute, McMaster University , Hamilton, Ontario, Canada .
authors.forename: Tony J
authors.initials: TJ
authors.lastname: Collins
authors.affiliation: 2 Department of Biological Sciences, Sunnybrook Research Institute , Toronto, Ontario, Canada .
authors.forename: Jarkko
authors.initials: J
authors.lastname: Ylanko
authors.affiliation: 3 School of Engineering Technology, McMaster University , Hamilton, Ontario, Canada .
authors.forename: Fei
authors.initials: F
authors.lastname: Geng
authors.affiliation: 2 Department of Biological Sciences, Sunnybrook Research Institute , Toronto, Ontario, Canada .
authors.forename: David W
authors.initials: DW
authors.lastname: Andrews
mesh_terms: D000165:Acridine Orange / Q000031:analogs & derivatives* / Q000032:analysis; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004396:Coloring Agents / Q000032:analysis*; D003513:Cycloheximide / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D015999:Multivariate Analysis; D013194:Staining and Labeling / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity
chemical_list: D004396:Coloring Agents; D014409:Tumor Necrosis Factor-alpha; C056258:N(10)-nonylacridine orange; D003513:Cycloheximide; D000165:Acridine Orange