Dataset Information

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.

SUBMITTER: Duellman SJ

PROVIDER: S-EPMC4605357 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

Duellman Sarah J SJ Zhou Wenhui W Meisenheimer Poncho P Vidugiris Gediminas G Cali James J JJ Gautam Prson P Wennerberg Krister K Vidugiriene Jolanta J

Assay and drug development technologies 20150918 8

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-de ...[more]

PMID: 26383544

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Project description:BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Dataset Information

Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

Publications

Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

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