Accurate Cytotoxicity and Proliferation Determination: Advantages of a High-Throughput Phenotypic Approach Over ATP Luminescence Assays.

Cell viability and proliferation assays are a fundamental tool in the drug discovery process and are used to evaluate both the antiproliferative potency and toxicity of compounds. Some lead discovery groups generate cell viability data for up to two million compounds per screen, so any method used to assess these parameters needs to deliver not only on data quality but also on throughput and assay cost per well. Most methods used to determine cell viability cannot deliver on all three of these requirements, so compromises have to be made. Here we show the development and implementation of a cost-effective, no-wash phenotypic assay to simultaneously report the number of cells, percentage of live cells, and cell cycle phase distribution as markers of proliferation and viability. We demonstrate that this assay can be applied to high-density plate formats and be imaged and analyzed in 8 min per plate on a laser scanning imaging cytometer. By comparing the drug-responses of several well-characterized anticancer drugs on HeLa cells, we highlight the key differences between the phenotypic assay and a commercial ATP luminescence detection system.

Advances in laser scanning imaging cytometry for high-content screening.

The advent of high-content screening more than a decade ago remodeled drug discovery workflows by recasting the role of cell-based approaches in target identification, primary screening, lead optimization, and toxicity. The ability to identify and quantify compound effects on multiple cellular functions allows for rapid characterization of chemical libraries. Laser scanning imaging cytometry (LSIC) is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using laser scanning excitation through a specialized F-theta scan lens. This configuration results in rapid whole well scanning and large depth of field. The recent availability of systems equipped with multiple sources of laser excitation and arrays of detectors for spectral analysis has significantly increased its applicability through enabling more fluorescent reagents and higher levels of multiplexing. LSIC is being used most extensively for phenotypic screening especially in areas such as cell health, RNA interference (RNAi) screening, and three-dimensional cell models. This review communicates advances in LSIC and how it is being applied by presenting an overview of the technology and a range of real-world case studies.

Determination of cell colony formation in a high-content screening assay.

The use of cell colony formation assays for research and clinical applications to assess the functional integrity of cells after in vitro manipulations is extensive. Key areas include hematopoietic stem cell research, cell transformation studies, and predicting the response of tumors to chemotherapeutic agents. Traditionally, enumeration of colonies has involved laborious and subjective counting by hand using a microscope. Here, laser scanning microplate cytometry has been used to provide an automated high-content readout of the effects of cytostatic agents on colony formation. This approach determines colony number through the application of a volume algorithm. Such an approach permits the differentiation of cytostatic effects where the number of colonies and size remains constant, and cytotoxic effects where the size and number may be reduced. Application of microplate cytometry thus offers significant benefits over alternative analytical methods in the search for novel chemotherapeutic agents.

Multiple cell lines using quantum dots.

The development of multiplexing capabilities and high-content readouts reporting individual cellular measurements enables assessment of biological variability on a single-cell basis, together with the evaluation of cell subpopulations within wells. A high-content screening multiplexed assay format allows additional information to be gained from a single assay. One such example is the ability to determine the effects of new chemical entities on different cell lines, tested in the same well. These assays, coupled with an appropriate automated cell-analysis platform, enable scalable screening of compound libraries for selectivity or toxicity. This approach can greatly increase screening efficiencies and enhance the amount of information achieved from a particular assay procedure, resulting in a significant reduction in the overall cost of a chemical compound library screen. By labeling live cells with Qtracker cell labeling kits and identifying cell proliferation using an Acumen Explorer microplate cytometer, we were able to determine the differential rates of cell proliferation of the individual cell lines in the same well over time. This method can extend to multiplexing more than two cell populations and to measure drug-induced differential changes in proliferation in a single-well assay on multiple cell lines.

Application of laser-scanning fluorescence microplate cytometry in high content screening.

The resolution of cell-based assays down to the cellular level has created new opportunities for the drug discovery process. Aptly named high content analysis, such an approach is enabling new methods of analysis for the broad range of therapeutic targets emerging in the post-genomics era, and offering alternative multiparametric readouts for some traditional analyses. Microplate cytometry is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using scanning laser excitation coupled to photomultiplier detection. This configuration results in area-based scanning across a large field of view, plus simultaneous detection of up to four emission colors for efficient multiplexing. Microplate cytometry is being used most extensively in the field of oncology research because of its usefulness for numerous applications, including protein kinase activity, cell cycle analysis, and cell colony formation. The review focuses on the Acumen Explorer microplate cytometer (TTP LabTech Ltd., Melbourn, Hertfordshire, UK), detailing the principal components of the instrument and providing an overview of its use in high content screening.