Flow Cytometry Multiplexing as a High Throughput Screening Method to Analyze Metabolic Activity for Pharmaceutical Drugs

Abstract

Abstract High throughput screening (HTS) is an extremely effective method that allows researchers to identify putative active compounds for therapeutics. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry is a fast and sensitive platform aiding in the study of protein-protein interactions, metabolic activities, as well as DNA content in a single or multi-parametric assay format. For this study, we screened several compound libraries and compared the results using a plate reader with flow cytometer multiplexing. Peripheral blood mononuclear (PBMCs) and Jurkat cells were used as cell models. Cells were cultured under standard conditions and either at normoxic (≤5%) or hyperoxic (≥19%) oxygen levels for varied lengths of time; 24, 48, or 72 hrs. Membrane integrity and metabolic activity were measured as an output for evaluating cellular viability. To further assess cellular activity, post-screening analysis was implemented to establish a drug dose-response and EC50s of “hits” from compound library. Screening analysis identified target drugs that were taken on to further tests using different mechanisms of action to assess compound toxicity. Results indicate that compound “hits” and potency differed in the screen depending on cell type, the mechanistic readout, length of compound exposure, and mode of readout used to perform the HTS experiments.