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A Fluorescence Microplate-Based Assay Workflow Enabling the Functional Characterization of Multi-Drug Resistance Transporters in Living CellsTélécharger
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March 31, 2011
Authors: Paul Held & Peter Banks, BioTek Instruments, Inc., Winooski, Vermont; Wayne Patton, Irina Lebedeva & Dee Shen, Enzo Life Sciences, Farmingdale, New York
An underlying mechanism for multi-drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents inside the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/ MXR (ABCG2) proteins. Various flow cytometry-based and microplatebased assays have facilitated determination of the functional expression levels of ABC transporters in living cells, but most dyes used as indicators (Rhodamine 123, DiOC2(3), calcein-AM) have limitations, since they fail to detect BCRP transporter protein. Dyes with broad transporter coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus generate a significant percentage of false negative results. We describe a novel fluorescent probe that serves as a substrate for all three ABC transporter types and can serve as an indicator of MDR in microplate-based assays using living cells. The probe exhibits fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multi-drug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, the workflow readily identifies functional efflux, as well as, defining the type of multi-drug resistance. We have optimized the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the assay, allowing, for the first time, easy quantitation of multi-drug resistance using a convenient fluorescence microplate-based HTS format that generates Z’ factor values greater than 0.5. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of MDR phenotypes.
EL406™ Combination Washer Dispenser