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A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication

Authors
  • Thayanithy, Venugopal1, 2
  • O’Hare, Patrick1
  • Wong, Phillip1
  • Zhao, Xianda3
  • Steer, Clifford J.4, 5
  • Subramanian, Subbaya3
  • Lou, Emil1, 6
  • 1 University of Minnesota, Department of Medicine, Division of Hematology, Oncology and Transplantation, Mayo Mail Code 480, 420 Delaware Street SE, Minneapolis, MN, 55455, USA , Minneapolis (United States)
  • 2 University of Minnesota Medical Center, Present Address: Molecular Diagnostics Laboratory, Fairview, 420 Delaware St SE, MMC 198, Minneapolis, MN, 55455, USA , Minneapolis (United States)
  • 3 University of Minnesota, Department of Surgery, Minneapolis, MN, 55455, USA , Minneapolis (United States)
  • 4 University of Minnesota, Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, Minneapolis, MN, 55455, USA , Minneapolis (United States)
  • 5 University of Minnesota, Department of Genetics, Cell Biology and Development, Minneapolis, MN, 55455, USA , Minneapolis (United States)
  • 6 University of Minnesota, Graduate Faculty, Department of Integrative Biology and Physiology, Minneapolis, MN, 55455, USA , Minneapolis (United States)
Type
Published Article
Journal
Cell Communication and Signaling
Publisher
BioMed Central
Publication Date
Nov 13, 2017
Volume
15
Issue
1
Identifiers
DOI: 10.1186/s12964-017-0201-2
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundTunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication.MethodsWe designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps.ResultsThe experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles.ConclusionsThis validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

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