Assessment of Drug Transporter Function Using Fluorescent Cell Imaging
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- Abstract
- Table of Contents
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- Literature Cited
Abstract
ATP?binding cassette (ABC) proteins, including the breast cancer resistance protein (BCRP) and multidrug resistance proteins (MDRs), actively transport structurally diverse chemicals from a number of tissues and are being increasingly cited as mediators of clinically relevant drug?drug interactions. The potential outcomes of concomitantly administering two drugs that interact at the same transporter include altered disposition and toxicity and/or efficacy of one or both of the drugs. Research demonstrating the role of transporters in clinical pharmacokinetics has shed light on the need for in vitro screening methods that detect drug?transporter interactions during preclinical development. This unit describes cell?based procedures for detecting functional inhibitors of BCRP and MDR1 by measuring fluorescent substrate accumulation in suspended cells using an automated cell counter, which offers convenience, sensitivity, and speed in measuring intracellular fluorescence and identifying new inhibitors. An alternative method is provided for making similar measurements using a spectrophotometer with fluorescence detection capabilities. Curr. Protoc. Toxicol . 57:21.12.1?21.12.15. © 2013 by John Wiley & Sons, Inc.
Keywords: ABC transporter; MDR1; BCRP; ABCB1; ABCG2
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Measurement of Transporter Function in Cells Overexpressing an ABC Transporter Using an Automated Fluorescent Cell Counter
- Alternate Protocol 1: Measurement of Transporter Function in Cells that Endogenously Express ABC Transporters Using an Automated Fluorescent Cell Counter
- Alternate Protocol 2: Measurement of Transporter Function in Cells Overexpressing an ABC Transporter Using a 96‐Well Plate Fluorescence Reader
- Commentary
- Literature Cited
- Figures
- Tables
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Measurement of Transporter Function in Cells Overexpressing an ABC Transporter Using an Automated Fluorescent Cell Counter
Materials
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Figure 23.6.1 Distribution of fluorescent substrate (FS) solution and medium in preparation for the addition of positive control inhibitor and various concentrations of test inhibitors in subsequent steps. View Image -
Figure 23.6.2 The layout for a 96‐well round‐bottom microtiter plate showing the placement of cell types and prepared solutions (100 µl). Abbreviations: FS, fluorescent substrate; FS + I, fluorescent substrate plus positive control inhibitor; no FS, no fluorescent substrate. View Image -
Figure 23.6.3 Cell loading procedure for imaging. This image demonstrates the addition of suspended cells (20 µl) into a counting chamber slide. It should be noted that a blue dye was used to visualize the loading. During a typical experiment, the solution will be colorless. View Image -
Figure 23.6.4 Example results with fluorescent substrates. Human embryonic kidney 293 cells stably transfected with human BCRP or MDR1 genes or empty vector plasmids were incubated with fluorescent substrates (BCRP, Hoechst 33342; MDR1, Rhodamine 123). Positive inhibitors (BCRP, Ko143; MDR1, PSC833) were used to block efflux of fluorescent substrates. (A ) The distribution of individual cell fluorescence. Each point represents the mean percent of cells ± SD exhibiting a quantity of fluorescence. (B ) Data (bar graphs) are presented as mean relative fluorescence ±SD normalized to cell size. View Image
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Literature Cited
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