title: Cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death.
abstract: Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beetle luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.
authors.affiliation: Promega Corporation, Madison, WI, USA. terry.riss@promega.com
authors.forename: Terry L
authors.initials: TL
authors.lastname: Riss
authors.forename: Richard A
authors.initials: RA
authors.lastname: Moravec
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Niles
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001681:Biological Assay; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003584:Cytological Techniques / Q000379:methods*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D051379:Mice; D009336:Necrosis; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D009842:Oligopeptides; C112765:tanespimycin; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; C078846:epoxomicin