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J Immunol Methods. 2016 Apr;431:1-10. doi: 10.1016/j.jim.2016.01.015. Epub 2016 Feb 2.

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

Author information

1
Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland.
2
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain.
3
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluis Companys 23, Barcelona 08010, Spain.
4
Universidad Carlos III of Madrid, Department of Bioengineering and Aerospace Engineering, Madrid 28911, Spain; Experimental Medicine and Surgery Unit, Instituto de Investigación Sanitaria del Hospital Gregorio Marañón, 28007 Madrid, Spain. Electronic address: jorge.ripoll@uc3m.es.
5
Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland. Electronic address: jstein@tki.unibe.ch.

Abstract

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.

KEYWORDS:

Light sheet fluorescence microscopy; Lymph nodes; T cell–DC interactions; Tissue clearing

PMID:
26844990
DOI:
10.1016/j.jim.2016.01.015
[Indexed for MEDLINE]

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