Assessment of Drug Transporter Function Using Fluorescent Cell Imaging

Kristin M. Bircsak1, Christopher J. Gibson1, Robert W. Robey2, Lauren M. Aleksunes3

1 Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey, 2 Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 3 Environmental and Occupational Health Sciences Institute, Piscataway, New Jersey
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 23.6
DOI:  10.1002/0471140856.tx2306s57
Online Posting Date:  September, 2013
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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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Table 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
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Basic Protocol 1: Measurement of Transporter Function in Cells Overexpressing an ABC Transporter Using an Automated Fluorescent Cell Counter

  • Fluorescent substrate, appropriate to the transporter, e.g., Rhodamine 123 (Sigma‐Aldrich) or Hoechst 33342 (Sigma‐Aldrich)
  • Positive control inhibitor, appropriate to the transporter, e.g., PSC833 (Xenotech) or Ko143 (Sigma‐Aldrich)
  • Dimethyl sulfoxide (DMSO)
  • Test inhibitors
  • Complete cell culture medium appropriate for cell line used
  • Cell dissociation medium: 0.25% (w/v) trypsin
  • Confluent cultures of cell lines overexpressing transporters of interest grown in 75‐cm2 cell culture flasks:
    • Laboratory‐generated cell lines transfected (e.g., using Lipofectamine) with plasmids containing ABC efflux transporters and empty vector controls (e.g., OriGene)
    • Commercially available cell lines, e.g., SB MDCKII BCRP and SB MDCKII MDR1, transfected cell lines; SB HL60 MRP1 and SB K562 MRP1 selected cell lines (Solvo Biotechnology)
  • Phosphate‐buffered saline (PBS; appendix 2A): sterilize by autoclaving or passing through a 0.2‐µm filter and chill in a 4°C refrigerator or on ice
  • 15‐ml tubes: sterile, amber‐colored, if available
  • 1.5‐ and 2‐ml microcentrifuge tubes, amber‐colored, if available
  • 2‐ to 25‐ml automatic serological pipettor (e.g., Easypet, Eppendorf)
  • Cellometer Vision automated cell counter and computer software (Nexcelom Bioscience)
  • 96‐well clear, round‐bottom microtiter plates with lids (e.g., Greiner Bio‐One Cellstar)
  • Microtiter plate centrifuge, set to 5°C
  • Biohazard waste container
  • Paper towels
  • 37°C, 5% CO 2 cell culture incubator
  • 100‐ to 1000‐µl eight‐channel automatic pipettor
  • 5‐ to 50‐µl eight‐channel manual pipettor
  • Aluminum foil
  • Cellometer counting chamber slides (Nexcelom Bioscience)
  • Cellometer Vision fluorescence optics modules (Nexcelom Bioscience)
    • VB‐450‐302 (Ex/Em: 375/450 nm for Hoechst 33342)
    • VB‐595‐502 (Ex/Em: 525/595 nm for Rhodamine 123)
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Literature Cited

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