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Label-Free Phenotype MicroArray™ Analysis of Cellular Energetics and Apoptotic Activity using Microplate Reading and Phase Contrast ImagingСкачать
Related Products: Cytation 5 Многорежимный фотометр-имиджер
February 03, 2015
Authors: Brad Larson, Leonie Rieger, BioTek Instruments, Inc. Winooski, VT USA; Jeffrey Travis, Larry Wiater, Biolog, Inc. Hayward, CA USA
Phenotypic screening, or the determination of the effects (phenotypes) that a molecule has on a cell, tissue, or whole organism, dates back to earliest drug discovery efforts. Due to advances in molecular biology and biochemistry in the 1990s, this approach was de-emphasized in favor of a more “reductionist” targetbased approach (Terstappen et al., 2007). Mounting evidence, however, indicates this shift not only failed to accelerate discovery of new first-in-class medicines, but has also led to higher attrition rates of new lead molecules (Swinney et al., 2013). Thus, a more balanced “holistic” approach, which incorporates both discovery methods, is increasingly being emphasized.
Phenotype MicroArrays™ is a unique label-free multiplexing technology for screening cell-based energetics that describe a variety of metabolically related phenotypes in a target cell model. By varying the available cellular nutrition sources, and measuring a cell’s metabolic activity in response to individual energy substrates present as a medium additive, information regarding metabolic pathway activity and sensitivity to chemicals can be elucidated. Differential responses to test molecules can be observed depending on available nutritional sources and the cellular genetic background. Exposure time of cells to each nutritional environment can also affect sensitivity. Through the incorporation of concomitant microscopic imaging, visual assessment of cellular health and morphology following adaptation to the carbon source can be carried out in a label-free manner, leading to a more complete interpretation of each data set.
Here we demonstrate a method to combine cellular metabolic Phenotype MicroArrays™ and automated, digital widefield phase contrast microscopy to screen differential nutritional pathway usage of test cells, in addition to potential pathway interruption by an apoptotic compound. Maintenance of a 5% CO2-95% air atmosphere at 37 ºC in the Cytation™ 5 plate chamber ensured sample integrity and enabled measurements of both cell morphology by image analysis and tetrazolium dye reduction rates, a reporter of cell energy production and cell viability in different nutritional environments. Gen5™ Software permitted multiplexed dye reduction kinetic analysis, in addition to apoptotic cells per well calculations. Results confirmed the validity of the combined methods to deliver a better understanding of the metabolic and toxic effects of pro-apoptotic molecules on target cell types.