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Development of a Live-Cell Based Reactive Oxygen Species (ROS) Assay for use in High-Content Screening of Drug Candidates Using The BioTek Synergy Mx Microplate Reader下載
April 21, 2010
Authors: Thomas A. Lyons, John J. Naleway, Marker Gene Technologies, Inc., University of Oregon Riverfront Research Park, Eugene, OR; Xavier F. Amouretti, Paul G. Held, BioTek Instruments, Inc., Winooski, VT
In healthy aerobic cells, reactive oxygen species (ROS) generation typically occurs at a controlled rate. But under stress conditions or by application of various drug candidates, ROS production can be greatly increased. The resulting changes in many cell components including DNA, proteins and lipids can influence cell viability, metabolism and growth. We have developed a live cell, high-throughput, microplate-based ROS assay that utilizes the cell permeable substrate, 2’,7’-dichlorofluorescin diacetate, a reliable fluorogenic marker for ROS detection. Upon enzyme activity, the highly fluorescent dye, 2’,7’-dichlorofluorescein is produced, with EX: 495 nm and EM: 530 nm. By using such a diacetate derivative, the assay can be efficiently adapted for intracellular applications, since the acetates help with effective loading into live cells. The acetates are then cleaved by endogenous intracellular esterases inside the cell, releasing the both adherent and non-adherent cell lines, upon treatment with the common chemotherapeutic drugs doxorubicin and idarubicin is presented. Dose response data is also compared with negative and positive controls (no drug or t-butyl hydroperoxide (TBHP) treatment). Methods for extrapolation of this assay for high-content screening applications are presented.
EL406 Combination Washer Dispenser