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A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.
Thayanithy, Venugopal; O'Hare, Patrick; Wong, Phillip; Zhao, Xianda; Steer, Clifford J; Subramanian, Subbaya; Lou, Emil.
Afiliação
  • Thayanithy V; Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, Minneapolis, MN, 55455, USA.
  • O'Hare P; Present Address: Molecular Diagnostics Laboratory, University of Minnesota Medical Center, Fairview, 420 Delaware St SE, MMC 198, Minneapolis, MN, 55455, USA.
  • Wong P; Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, Minneapolis, MN, 55455, USA.
  • Zhao X; Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, Minneapolis, MN, 55455, USA.
  • Steer CJ; Department of Surgery, University of Minnesota, Minneapolis, MN, 55455, USA.
  • Subramanian S; Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, MN, 55455, USA.
  • Lou E; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
Cell Commun Signal ; 15(1): 46, 2017 Nov 13.
Article em En | MEDLINE | ID: mdl-29132390
BACKGROUND: Tunneling 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: This 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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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Comunicação Celular / Nanotubos / Espaço Extracelular / Exossomos Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Comunicação Celular / Nanotubos / Espaço Extracelular / Exossomos Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article