title: Isolation of brain-infiltrating leukocytes.
abstract: We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 μm, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1(+), CD11b(+) and F4/80(+)cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.
authors.affiliation: Department of Neurology, Mayo Clinic College of Medicine, USA.
authors.forename: Reghann G
authors.initials: RG
authors.lastname: LaFrance-Corey
authors.forename: Charles L
authors.initials: CL
authors.lastname: Howe
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology* / Q000276:immunology / Q000473:pathology / Q000821:virology; D018188:Cardiovirus Infections / Q000276:immunology / Q000473:pathology / Q000821:virology; D003584:Cytological Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D005625:Frontal Lobe / Q000166:cytology / Q000276:immunology / Q000473:pathology / Q000821:virology; D016130:Immunophenotyping / Q000379:methods; D007962:Leukocytes / Q000166:cytology* / Q000276:immunology / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D017945:Theilovirus