A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication
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 T...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-11-01
|
| Series: | Cell Communication and Signaling |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12964-017-0201-2 |
| _version_ | 1819139112979922944 |
|---|---|
| author | Venugopal Thayanithy Patrick O’Hare Phillip Wong Xianda Zhao Clifford J. Steer Subbaya Subramanian Emil Lou |
| author_facet | Venugopal Thayanithy Patrick O’Hare Phillip Wong Xianda Zhao Clifford J. Steer Subbaya Subramanian Emil Lou |
| author_sort | Venugopal Thayanithy |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T11:17:29Z |
| format | Article |
| id | doaj.art-23136548187c4ce79af6af48ef91951c |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-12-22T11:17:29Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-23136548187c4ce79af6af48ef91951c2022-12-21T18:27:58ZengBMCCell Communication and Signaling1478-811X2017-11-0115111610.1186/s12964-017-0201-2A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communicationVenugopal Thayanithy0Patrick O’Hare1Phillip Wong2Xianda Zhao3Clifford J. Steer4Subbaya Subramanian5Emil Lou6Department of Medicine, Division of Hematology, Oncology and Transplantation, University of MinnesotaDepartment of Medicine, Division of Hematology, Oncology and Transplantation, University of MinnesotaDepartment of Medicine, Division of Hematology, Oncology and Transplantation, University of MinnesotaDepartment of Surgery, University of MinnesotaDepartment of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of MinnesotaDepartment of Surgery, University of MinnesotaDepartment of Medicine, Division of Hematology, Oncology and Transplantation, University of MinnesotaAbstract 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.http://link.springer.com/article/10.1186/s12964-017-0201-2Tunneling nanotubesMembrane nanotubesIntercellular transferIntercellular communicationTranswell assayExosomes |
| spellingShingle | Venugopal Thayanithy Patrick O’Hare Phillip Wong Xianda Zhao Clifford J. Steer Subbaya Subramanian Emil Lou A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication Cell Communication and Signaling Tunneling nanotubes Membrane nanotubes Intercellular transfer Intercellular communication Transwell assay Exosomes |
| title | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication |
| title_full | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication |
| title_fullStr | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication |
| title_full_unstemmed | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication |
| title_short | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication |
| title_sort | transwell assay that excludes exosomes for assessment of tunneling nanotube mediated intercellular communication |
| topic | Tunneling nanotubes Membrane nanotubes Intercellular transfer Intercellular communication Transwell assay Exosomes |
| url | http://link.springer.com/article/10.1186/s12964-017-0201-2 |
| work_keys_str_mv | AT venugopalthayanithy atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT patrickohare atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT phillipwong atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT xiandazhao atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT cliffordjsteer atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT subbayasubramanian atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT emillou atranswellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT venugopalthayanithy transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT patrickohare transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT phillipwong transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT xiandazhao transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT cliffordjsteer transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT subbayasubramanian transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication AT emillou transwellassaythatexcludesexosomesforassessmentoftunnelingnanotubemediatedintercellularcommunication |