title: Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.
abstract: Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.
authors.affiliation: 1 Research and Development , Promega Corporation, Madison, Wisconsin.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Duellman
authors.affiliation: 2 Promega Biosciences, LLC , San Luis Obispo, California.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhou
authors.affiliation: 2 Promega Biosciences, LLC , San Luis Obispo, California.
authors.forename: Poncho
authors.initials: P
authors.lastname: Meisenheimer
authors.affiliation: 1 Research and Development , Promega Corporation, Madison, Wisconsin.
authors.forename: Gediminas
authors.initials: G
authors.lastname: Vidugiris
authors.affiliation: 1 Research and Development , Promega Corporation, Madison, Wisconsin.
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
authors.affiliation: 3 Institute for Molecular Medicine Finland, University of Helsinki , Helsinki, Finland .
authors.forename: Prson
authors.initials: P
authors.lastname: Gautam
authors.affiliation: 3 Institute for Molecular Medicine Finland, University of Helsinki , Helsinki, Finland .
authors.forename: Krister
authors.initials: K
authors.lastname: Wennerberg
authors.affiliation: 1 Research and Development , Promega Corporation, Madison, Wisconsin.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Vidugiriene
mesh_terms: D000970:Antineoplastic Agents / Q000032:analysis* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D003199:Computer Systems; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D020014:K562 Cells; D008163:Luminescent Measurements / Q000379:methods*; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D054852:Small Molecule Libraries