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Ensemble and Single Quantum Dot Fluorescence Methods in Neurotransmitter Transporter Research

  • Oleg Kovtun
  • Sandra J. RosenthalEmail author
Protocol
Part of the Neuromethods book series (NM, volume 118)

Abstract

Subcellular localization and trafficking of neurotransmitter transporter (NTT) proteins is increasingly recognized to play a critical role in transporter-mediated neurotransmitter signaling and its regulation. To fully understand the molecular mechanisms underlying transporter regulation, it is essential to be able to visualize NTTs both at the population and single-molecule levels using advanced imaging techniques. Here, we describe three fluorescence-based methods that have been successfully applied to measure spatiotemporal changes in NTT localization and to establish dynamic imaging of individual NTT molecules using the ligand-conjugated quantum dot (QD) approach. First, we discuss how to label and image membrane NTTs in live cells using QD probes in conjunction with ensemble fluorescence microscopy. Second, we present a more quantitative, flow cytometry-based approach, particularly useful for assessing transporter internalization and recycling. Third, we describe a single-molecule microscopy labeling protocol for determining the mobility of QD-bound transporters at the plasma membrane of live cells.

Key words

Quantum dot Biological labeling Neurotransmitter transporter Confocal fluorescence microscopy Flow cytometry Biotinylated ligand Single-molecule imaging 

Notes

Acknowledgements

The authors wish to thank Prof. Randy D. Blakely, Dr. Jerry C. Chang, and Dr. Ian D. Tomlinson for all the helpful discussions and suggestions. This work was supported by grants from National Institutes of Health EB003728 to S.J.R. O.K. would also like to acknowledge Vanderbilt Institute for Nanoscale Science and Engineering (VINSE) fellowship.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of ChemistryVanderbilt UniversityNashvilleUSA
  2. 2.Department of PharmacologyVanderbilt UniversityNashvilleUSA
  3. 3.Department of Chemical and Biomolecular EngineeringVanderbilt UniversityNashvilleUSA
  4. 4.Department of Physics and AstronomyVanderbilt UniversityNashvilleUSA
  5. 5.Vanderbilt Institute of Nanoscale Science and EngineeringVanderbilt UniversityNashvilleUSA
  6. 6.Materials Science and Technology DivisionOak Ridge National LaboratoryOak RidgeUSA

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