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Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1)
Authors:Edit Szabó  ,Anna Kulin,Bá  lint Jezsó  ,Nó  ra Kucsma,Balá  zs Sarkadi,Gyö  rgy Vá  rady
Affiliation:1.Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary;2.Doctoral School of Molecular Medicine, Semmelweis University, 1085 Budapest, Hungary;3.Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary
Abstract:The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.
Keywords:ABC transporter   ABCB1   P-glycoprotein   functional biology   multidrug transporter   TO-PRO-3   TO-PRO-1   cyanine dye
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