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

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Purinergic Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2041))

Abstract

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.

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Authors and Affiliations

  1. Visual Neuroscience Laboratory, Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, VIC, Australia

    Kirstan A. Vessey, Tracy Ho, Andrew I. Jobling, Anna Y. Wang & Erica L. Fletcher

Authors
  1. Kirstan A. Vessey
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  2. Tracy Ho
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  3. Andrew I. Jobling
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  4. Anna Y. Wang
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  5. Erica L. Fletcher
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Corresponding author

Correspondence to Kirstan A. Vessey .

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Editors and Affiliations

  1. BioMedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain

    Pablo Pelegrín

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Vessey, K.A., Ho, T., Jobling, A.I., Wang, A.Y., Fletcher, E.L. (2020). Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging. In: Pelegrín, P. (eds) Purinergic Signaling. Methods in Molecular Biology, vol 2041. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9717-6_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9717-6_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9716-9

  • Online ISBN: 978-1-4939-9717-6

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