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Published on: May 10, 2016
HighVia-A Flexible Live-Cell High-Content Screening Pipeline to Assess Cellular Toxicity
Alison Howarth1,2, Martin Schröder3,2, Raquel C Montenegro4,5,2
1Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.
Abstract:
High-content screening to monitor disease-modifying phenotypes upon small-molecule addition has become an essential component of many drug and target discovery platforms. One of the most common phenotypic approaches, especially in the field of oncology research, is the assessment of cell viability. However, frequently used viability readouts employing metabolic proxy assays based on homogeneous colorimetric/fluorescent reagents are one-dimensional, provide limited information, and can in many cases yield conflicting or difficult-to-interpret results, leading to misinterpretation of data and wasted resources.The resurgence of high-content, phenotypic screening has significantly improved the quality and breadth of cell viability data, which can be obtained at the very earliest stages of drug and target discovery. Here, we describe a relatively inexpensive, high-throughput, high-content, multiparametric, fluorescent imaging protocol using a live-cell method of three fluorescent probes (Hoechst, Yo-Pro-3, and annexin V), that is amenable to the addition of further fluorophores. The protocol enables the accurate description and profiling of multiple cell death mechanisms, including apoptosis and necrosis, as well as accurate determination of compound IC50, and has been validated on a range of high-content imagers and image analysis software. To validate the protocol, we have used a small library of approximately 200 narrow-spectrum kinase inhibitors and clinically approved drugs. This fully developed, easy-to-use pipeline has subsequently been implemented in several academic screening facilities, yielding fast, flexible, and rich cell viability data for a range of early-stage high-throughput drug and target discovery programs.
Insights
This study introduces a high-content, multiparametric imaging protocol for precise cell viability assessment in drug discovery. The method accurately profiles cell death mechanisms and determines compound efficacy, improving early-stage research.
Area of Science:
- Biotechnology
- Drug Discovery
- Cell Biology
Background:
- High-content screening is crucial for monitoring disease-modifying phenotypes in drug discovery.
- Traditional cell viability assays often provide limited, one-dimensional data, leading to misinterpretation.
- High-content, phenotypic screening offers improved quality and breadth of cell viability data.
Purpose of the Study:
- To develop an inexpensive, high-throughput, multiparametric fluorescent imaging protocol for cell viability assessment.
- To enable accurate profiling of multiple cell death mechanisms (apoptosis, necrosis).
- To facilitate precise determination of compound IC50 values in early-stage drug discovery.
Main Methods:
- Utilized a live-cell, multiparametric fluorescent imaging protocol with Hoechst, Yo-Pro-3, and annexin V probes.
- The protocol is amenable to additional fluorophores for enhanced analysis.
- Validated on various high-content imagers and image analysis software using kinase inhibitors and approved drugs.
Main Results:
- The protocol accurately describes and profiles multiple cell death mechanisms.
- Accurate determination of compound IC50 values was achieved.
- Validated on a library of ~200 kinase inhibitors and clinically approved drugs.
Conclusions:
- The developed pipeline provides fast, flexible, and rich cell viability data for early-stage drug discovery.
- This method enhances the accuracy and depth of cell viability analysis.
- Successfully implemented in academic screening facilities for drug and target discovery programs.
