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Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro

Nature Protocols volume 10, pages 2016–2026 (2015)Cite this article

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Abstract

We have recently demonstrated that brain endothelial cells cross-present parasite antigen during mouse experimental cerebral malaria (ECM). Here we describe a 2-d protocol to detect cross-presentation by isolating the brain microvessels and incubating them with a reporter cell line that expresses lacZ upon detection of the relevant peptide–major histocompatibility complex. After X-gal staining, a typical positive result consists of hundreds of blue spots, compared with fewer than 20 spots from a naive brain. The assay is generalizable to other disease contexts by using reporter cells that express appropriate specific T cell receptors. Also described is the protocol for culturing endothelial cells from brain microvessels isolated from naive mice. After 7–10 d, an in vitro cross-presentation assay can be performed by adding interferon-γ, antigen (e.g., Plasmodium berghei–infected red blood cells) and reporter cells in sequence over 3 d. This is useful for comparing different antigen forms or for probing the effects of various interventions.

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Figure 1: Schematic of the cross-presentation assays.
Figure 2: Photographs of brain microvessel isolation steps.
Figure 3: Typical results of the ex vivo brain microvessel cross-presentation assay using LR-BSL8.4a reporter cells.
Figure 4: Light microscope images of various stages of MBEC culture.

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Acknowledgements

This work was funded by an intramural grant from Singapore's Agency for Science, Technology and Research. S.Y.G. and C.M.P. were supported by postgraduate scholarships from the Yong Loo Lin School of Medicine, National University of Singapore. We thank N. Shastri (University of California, Berkeley) for providing the BWZ.36/CD8α cells.

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Authors and Affiliations

  1. Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Shanshan W Howland, Sin Yee Gun, Carla Claser, Chek Meng Poh & Laurent Rénia

  2. Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Sin Yee Gun, Chek Meng Poh & Laurent Rénia

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  1. Shanshan W Howland
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Contributions

S.W.H. conceived the methods and C.C., S.Y.G. and C.M.P. helped optimize the protocols. L.R. supervised the project. S.W.H. and L.R. wrote and edited the manuscript.

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Correspondence to Shanshan W Howland or Laurent Rénia.

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Howland, S., Gun, S., Claser, C. et al. Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro. Nat Protoc 10, 2016–2026 (2015). https://doi.org/10.1038/nprot.2015.129

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