Confocal imaging of neuropeptide Y-pHluorin: A technique to visualize insulin granule exocytosis in intact murine and human islets

Madina Makhmutova, Tao Liang, Herbert Gaisano, Diego A Caicedo-Vierkant, Joana Almaca

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 μm of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

Original languageEnglish (US)
Article numbere56089
JournalJournal of Visualized Experiments
Volume2017
Issue number127
DOIs
StatePublished - Sep 13 2017

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Keywords

  • Adenovirus
  • Cellular biology
  • Exocytosis
  • Insulin granule
  • Issue 127
  • Neuropeptide Y
  • Pancreatic islets
  • PHluorin
  • Pulsatile secretion

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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