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.
Highlights
Cell viability is a universal endpoint that is measured in many drug discovery efforts across a variety of disease models
RealTime-Glo MT Cell Viability Assay, CellTiter-Glo, CellTiter-Fluor, and Caspase-Glo 3/7 were obtained from protease was measured using the CellTiter-Fluor Assay (Promega) Corporation
Assay Principle and Real-Time Applications The real-time cell viability assay consists of two components, a luciferase enzyme and a prosubstrate (Fig. 1A), which are added to the cell culture media
Summary
Cell viability is a universal endpoint that is measured in many drug discovery efforts across a variety of disease models. Each biomarker provides advantages based on the experimental goals, but most assays are used in endpoint formats that destroy the cells and are incompatible with the kinetic or real-time analysis of compound toxicity. Because the assay was well tolerated by cells and the assay chemistry is compatible with many different multiplexes, including other luminescent assays without the need for spectral filters, we were able to establish the optimal time to multiplex a luminescent apoptosis assay to capture the caspase activation window and avoid falsenegative results. This multiplex provided more meaningful data through interrogation of the same experimental samples, thereby providing an internally controlled dataset
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