Application Note: Quantification of Cytotoxicity Using the Incucyte® Cytotoxicity Assay
Cell death is a dynamic process, and the monitoring of cell cytotoxicity is critical in many therapeutic areas, such as oncology. Exposure of cells to a cytotoxic stimulus may compromise metabolic activity, inhibit cell growth or ultimately, via complex biological pathways such as necrosis or apoptosis, result in cell death.
Regardless of the mechanism, cytotoxic compounds often compromise cell membrane integrity, and once a cell irreversibly loses its membrane integrity, it is destined to die.
Assays designed to measure cytotoxicity in vitro are used to predict tissue-specific toxicity or identify and classify leads for anti-cancer therapies. Multiplexed, high-throughput screening (HTS) cytotoxicity assays measuring a variety of different readouts are being employed to assess the cytotoxicity of compounds during pharmacological investigations in early drug development.
Although there are several multiplexed cytotoxicity assays involving measurement of membrane integrity, which can simultaneously measure multiple indicators of in vitro cytotoxicity, they typically assess a single time point and are unable to assess the biological activity over time.
To kinetically measure cell membrane integrity, live-cell analysis methods have been developed. However, optimized technology and reagents are required for flexible and accurate assessment. Use of the Incucyte® Live-Cell Analysis System in conjunction with Incucyte® Cytotox Dyes, as a live-cell kinetic assay for the measurement of cytotoxicity, demonstrates quantitative and reproducible detection of cell permeability - a hallmark of cell death.
This approach also provides the ability to monitor morphological changes in parallel with quantification, in physiologically relevant conditions. The combination of Incucyte® Cytotox Dyes with Incucyte® Nuclight Reagents or cells lines, enables multiplexed measurements of proliferation and cytotoxicity.
In addition, Incucyte® Cell-by-Cell Analysis Software provides individual cell segmentation and classification based on fluorescence, allowing for additional parameters such as the cytotoxic or apoptotic index, to be calculated and facilitates assessment of drug mechanism actions on cell populations.
In this application note, we describe the utility of Incucyte® Cytotoxicity Assays, encompassing no-wash, mix-and-read reagents, and integrated image-based analysis tools that enable kinetic quantification of cytotoxicity.
Validation data highlighted in this Application Note include:
- Quantification of Cytotoxicity
- Profiling of Single Compounds Across Multiple Cancer Cell Types
- Multiplexed Measurements of Proliferation and Cytotoxicity
- Subpopulation Analysis of Cytotoxic and Cytostatic Treatment Effects
