Journal
NATURE PROTOCOLS
Volume 10, Issue 12, Pages 2016-2026Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.129
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Funding
- intramural grant from Singapore's Agency for Science, Technology and Research
- Yong Loo Lin School of Medicine, National University of Singapore
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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-gamma, 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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