title: Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro.
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.
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Shanshan W
authors.initials: SW
authors.lastname: Howland
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.|Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Sin Yee
authors.initials: SY
authors.lastname: Gun
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Carla
authors.initials: C
authors.lastname: Claser
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.|Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Chek Meng
authors.initials: CM
authors.lastname: Poh
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.|Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Laurent
authors.identifier: http://orcid.org/0000-0003-0349-1557
authors.initials: L
authors.lastname: Rénia
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D001921:Brain / Q000098:blood supply / Q000166:cytology / Q000276:immunology*; D018929:Cell Culture Techniques; D002460:Cell Line; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000166:cytology / Q000276:immunology*; D008285:Major Histocompatibility Complex; D008288:Malaria / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000166:cytology / Q000276:immunology*; D010962:Plasmodium berghei / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology
chemical_list: D011948:Receptors, Antigen, T-Cell