Published: Vol 8, Iss 5, Mar 5, 2018 DOI: 10.21769/BioProtoc.2757 Views: 6705
Reviewed by: Ivan ZanoniYang FuMeenal Sinha
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
An Automated Imaging Method for Quantification of Changes to the Endomembrane System in Mammalian Spheroid Models
Margaritha M. Mysior and Jeremy C. Simpson
Jun 5, 2025 636 Views
PI(4,5)P2 Imaging Using a GFP Reporter in Living Cells
Mariam Alkandari [...] Mahtab Tavasoli
Jun 5, 2025 722 Views
Quantifying Intracellular Distributions of HaloTag-Labeled Proteins With SDS-PAGE and Epifluorescence Microscopy
Julia Shangguan and Ronald S. Rock
Jul 20, 2025 1119 Views
Abstract
Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T cells. Additional cell types might also be found in the PVS and initiate interactions with exiting T cells. The exact content of the PVS, and the processes within, are not well studied. To distinguish vascular from resident cells within various tissues by flow cytometry, intravenous (i.v.) labeling is becoming a commonly employed method. We recently used anti-CD45.2 antibodies and magnetic enrichment to further evaluate this technique, and compared labeled and unlabeled cells in the thymus and blood. This assay can be used to specifically investigate hematopoietic cell subsets within the PVS of the thymus.
Keywords: Perivascular spacesBackground
Immature thymocytes undergo a series of maturation steps, including positive and negative selection, which eliminate the majority of developing T cells. The resulting mature T cell pool is thereby shaped towards a higher proportion of beneficial clones and a reduced proportion of dangerous self-reactive clones. The thymus also produces less abundant T cell subsets that generally act to maintain immune system, tissue, and metabolic homeostasis, including: TCRγδ cells, regulatory T (Treg) cells, natural killer T (NKT) cells, intraepithelial lymphocyte (IEL) precursors, and mucosal associated invariant T (MAIT) cells. Mature thymocytes poised to emigrate into the periphery upregulate expression of the receptor (S1PR1) that recognizes sphingosine-1 phosphate (S1P), a lipid molecule present at high concentrations in the blood. S1PR1+ T cells migrate along an S1P gradient and wind up in vascular circulation.
The thymic perivascular spaces (PVS) are basement membrane-separated compartments between the parenchyma and the vasculature. They are thought to facilitate trafficking of cells, especially mature T cells emigrating from the thymus (Mori et al., 2007; Weinreich and Hogquist, 2008; Zachariah and Cyster, 2010). The exact content of the PVS is not well characterized yet, and could include antigen presenting cells such as dendritic cells and macrophages, that carry antigens not normally expressed in the thymus, into this tissue to contribute to thymocyte selection processes.
Intravenous (i.v.) labeling is a technique commonly used to distinguish vasculature-associated circulating cells from those residing within tissues at the time of analysis (Anderson et al., 2014). Cyster and colleagues have used this approach to identify CD4+ emigrating T cells within the PVS, and showed that upon tail-vein injection of CD4-labeling antibody, CD4 T cells in the PVS are positively labeled within 3 min (Zachariah and Cyster, 2010). We sought to establish whether thymic precursors of CD8αα IEL, an agonist selected T cell subset that downregulates both CD4 and CD8 expression during thymic maturation, can also be found in the PVS. In order to do so, we adapted the i.v. labeling approach, using phycoerythrin (PE)-conjugated anti-CD45.2 (for C57BL/6 mice). As CD45 is not T cell specific, but is expressed by hematopoietic cells in general, various cells can be identified within the i.v.-labeled (IV+) fraction. Furthermore, we combined this with magnetic enrichment for the PE-conjugated antibodies. This allowed us to more closely evaluate the perivascular contents.
Materials and Reagents
Equipment
Software
Procedure
Note: All animal procedures must be approved by your institution’s ethics committee.
Data analysis
Notes
Recipes
Acknowledgments
We would like to thank Rebecca Kummer for assistance with some i.v. labeling experiments, and Oscar Salgado Barrero for assistance with taking photographs. This work was supported by NIH grants R37 AI39560 and PO1 AI35296 to K.A.H. The authors have no competing financial interests.
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Ruscher, R. and Hogquist, K. A. (2018). Intravenous Labeling and Analysis of the Content of Thymic Perivascular Spaces. Bio-protocol 8(5): e2757. DOI: 10.21769/BioProtoc.2757.
Category
Immunology > Animal model > Mouse
Cell Biology > Cell imaging > Fluorescence
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link