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Deprecated: Implicit conversion from float 247.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Cell+Commun+Signal 2017 ; 15 (ä): ä Nephropedia Template TP
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A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication #MMPMID29132390
Thayanithy V; O?Hare P; Wong P; Zhao X; Steer CJ; Subramanian S; Lou E
Cell Commun Signal 2017[]; 15 (ä): ä PMID29132390show ga
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. Electronic supplementary material: The online version of this article (10.1186/s12964-017-0201-2) contains supplementary material, which is available to authorized users.