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Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Forster, S, Thumser, AE, Hood, SR and Plant, NJ (2012) Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays PLoS One, 7 (3), e33253.

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Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex=505nm, λem=525nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine > Department of Biochemical Sciences
Authors :
Forster, S
Thumser, AE
Hood, SR
Plant, NJ
Date : 28 March 2012
DOI : 10.1371/journal.pone.0033253
Uncontrolled Keywords : IVIVE, Drug Transporter, MDR1, pGP
Additional Information : © 2012 Forster et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Depositing User : Symplectic Elements
Date Deposited : 09 May 2012 09:33
Last Modified : 31 Oct 2017 14:32

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