A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.
Cell Commun Signal
; 15(1): 46, 2017 Nov 13.
Article
em En
| MEDLINE
| ID: mdl-29132390
ABSTRACT
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.Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Comunicação Celular
/
Nanotubos
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Espaço Extracelular
/
Exossomos
Limite:
Humans
Idioma:
En
Revista:
Cell Commun Signal
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos