Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at p...

Full description

Bibliographic Details
Authors: Abe, Jun, Ozga, Aleksandra J., Swoger, Jim, Sharpe, James, Ripoll, Jorge, Stein, Jens V.
Format: article
Status:Versión aceptada para publicación
Publication Date:2016
Country:España
Institution:Universitat Pompeu Fabra
Repository:Repositorio Digital de la UPF
OAI Identifier:oai:repositori.upf.edu:10230/27845
Online Access:http://hdl.handle.net/10230/27845
http://dx.doi.org/10.1016/j.jim.2016.01.015
Access Level:Open access
Keyword:Cèl·lules
Llum
Nodes limfàtics
Microscòpia de fluorescència
id ES_fdc4b7015b10941b3a6f0b24712e8f2a
oai_identifier_str oai:repositori.upf.edu:10230/27845
network_acronym_str ES
network_name_str España
repository_id_str
spelling Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodesAbe, JunOzga, Aleksandra J.Swoger, JimSharpe, JamesRipoll, JorgeStein, Jens V.Cèl·lulesLlumNodes limfàticsMicroscòpia de fluorescènciaReactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.This work was funded by Swiss National Foundation grant 31003A_135649 (to JVS), Sinergia grant CR23I3_156234 and CRSII3_141918 (to JSh and JVS), EC FP7 Marie Curie RG grant 276702 (to JVS), and a Novartis Research (NRG-2011-0341) grant (to AO). JA was supported by Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science. JR acknowledges support from the EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and MINECO grant FIS2013-41802-R MESO-IMAGING. JSw and JSh acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. The SPIM setup was financed by a grant of the Pierre Mercier Foundation.Elsevier20172016info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10230/27845http://dx.doi.org/10.1016/j.jim.2016.01.015reponame:Repositorio Digital de la UPFinstname:Universitat Pompeu FabraInglésJournal of Immunological Methods. 2016 Apr;431:1-10info:eu-repo/grantAgreement/EC/FP7/276702© Elsevier http://dx.doi.org/10.1016/j.jim.2016.01.015info:eu-repo/semantics/openAccessoai:repositori.upf.edu:10230/278452026-06-12T07:21:37Z
dc.title.none.fl_str_mv Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
title Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
spellingShingle Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
Abe, Jun
Cèl·lules
Llum
Nodes limfàtics
Microscòpia de fluorescència
title_short Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
title_full Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
title_fullStr Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
title_full_unstemmed Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
title_sort Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
dc.creator.none.fl_str_mv Abe, Jun
Ozga, Aleksandra J.
Swoger, Jim
Sharpe, James
Ripoll, Jorge
Stein, Jens V.
author Abe, Jun
author_facet Abe, Jun
Ozga, Aleksandra J.
Swoger, Jim
Sharpe, James
Ripoll, Jorge
Stein, Jens V.
author_role author
author2 Ozga, Aleksandra J.
Swoger, Jim
Sharpe, James
Ripoll, Jorge
Stein, Jens V.
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Cèl·lules
Llum
Nodes limfàtics
Microscòpia de fluorescència
topic Cèl·lules
Llum
Nodes limfàtics
Microscòpia de fluorescència
description Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.
publishDate 2016
dc.date.none.fl_str_mv 2016
2017
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10230/27845
http://dx.doi.org/10.1016/j.jim.2016.01.015
url http://hdl.handle.net/10230/27845
http://dx.doi.org/10.1016/j.jim.2016.01.015
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Journal of Immunological Methods. 2016 Apr;431:1-10
info:eu-repo/grantAgreement/EC/FP7/276702
dc.rights.none.fl_str_mv © Elsevier http://dx.doi.org/10.1016/j.jim.2016.01.015
info:eu-repo/semantics/openAccess
rights_invalid_str_mv © Elsevier http://dx.doi.org/10.1016/j.jim.2016.01.015
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Repositorio Digital de la UPF
instname:Universitat Pompeu Fabra
instname_str Universitat Pompeu Fabra
reponame_str Repositorio Digital de la UPF
collection Repositorio Digital de la UPF
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869425597225107456
score 15,812455

Similar Items