Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging

  • Kirstan A. VesseyEmail author
  • Tracy Ho
  • Andrew I. Jobling
  • Anna Y. Wang
  • Erica L. Fletcher
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)


Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

Key words

Retina VNUT Purine P2X7 Adenosine Purinergic Solute carrier 17, member 9 (SLC17A9Dopamine Dopaminergic 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Kirstan A. Vessey
    • 1
    Email author
  • Tracy Ho
    • 1
  • Andrew I. Jobling
    • 1
  • Anna Y. Wang
    • 1
  • Erica L. Fletcher
    • 1
  1. 1.Visual Neuroscience Laboratory, Department of Anatomy and NeuroscienceThe University of MelbourneParkvilleAustralia

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