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Abstract
Abstract High throughput screening (HTS) is extremely effective method for allowing researchers to identify putative compounds of interest which play a role in their area of cellular research. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry not only allows a researcher to study drug effectiveness towards different cell types but also can be used to analyze protein-protein interactions, metabolic activities, as well as DNA/mRNA content in a single or multiplexed assay format. In this study, we screened the MicroSource Discovery System Killer Plate compound library and compared HTS using a plate reader with flow cytometer multiplexing. Jurkat and Ramos cells, T and B lymphocyte cell types respectively, were used as cell models and cultured under standard conditions and screened either at hypoxic (1%) or hyperoxic (19%) oxygen levels at varied lengths of time (24, 48, or 72 hrs). To assess compound veracity, post-screening analysis was implemented to establish EC50s of “hits” from the compound library. Membrane integrity and metabolic activity were measured as an initial screening output for evaluating cellular viability. Using the multiplexed capabilities of flow cytometry, we performed secondary and tertiary assays to further characterize the possible mechanism of action by analysis of cell cycle, specific RNA expression levels, oxidative stress, and caspase activation across differing concentrations of the compounds. This method and analysis highlights the complicated nature of assessing toxicity in cellular screening assays and the advantage of Flow Cytometry in particular for characterizing cell health.
Published Version
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