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

Cell Communication and Signaling

Methodology

Clifford J. Steer¹  Phillip Wong²  Patrick O’Hare²  Emil Lou³  Venugopal Thayanithy⁴  Xianda Zhao⁵  Subbaya Subramanian⁵ 

[1] Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, 55455, Minneapolis, MN, USA;Department of Genetics, Cell Biology and Development, University of Minnesota, 55455, Minneapolis, MN, USA;Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, 55455, Minneapolis, MN, USA;Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, 55455, Minneapolis, MN, USA;Graduate Faculty, Department of Integrative Biology and Physiology, University of Minnesota, 55455, Minneapolis, MN, USA;Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street SE, 55455, Minneapolis, MN, USA;Present Address: Molecular Diagnostics Laboratory, University of Minnesota Medical Center, Fairview, 420 Delaware St SE, MMC 198, 55455, Minneapolis, MN, USA;Department of Surgery, University of Minnesota, 55455, Minneapolis, MN, USA;

关键词: Tunneling nanotubes;    Membrane nanotubes;    Intercellular transfer;    Intercellular communication;    Transwell assay;    Exosomes;    Microvesicles;    Extracellular vesicles;   

DOI  :  10.1186/s12964-017-0201-2

 received in 2017-09-21, accepted in 2017-10-30,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s). 2017