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A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3′,5′-Cyclic Adenosine Monophosphate Efflux

  • Dominique Perez
  • Peter C. Simons
  • Yelena Smagley
  • Larry A. SklarEmail author
  • Alexandre Chigaev
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1439)

Abstract

Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3′,5′-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).

Key words

Cyclic AMP Fluorescent substrates High-throughput flow cytometry Efflux inhibitors ABC transporters 

Notes

Acknowledgments

This work was supported by The Oxnard Foundation, University of New Mexico Clinical & Translational Science Center Pilot Award 1UL1RR031977, and New Mexico Cancer Nanotechnology Training Center grant R25CA153825.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Dominique Perez
    • 1
  • Peter C. Simons
    • 1
  • Yelena Smagley
    • 1
  • Larry A. Sklar
    • 1
    Email author
  • Alexandre Chigaev
    • 1
  1. 1.University of New Mexico Cancer CenterCenter for Molecular Discovery, University of New MexicoAlbuquerqueUSA

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