Isolation of brain-infiltrating leukocytes

Reghann G. Lafrance-Corey, Charles L Howe

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere2747
JournalJournal of Visualized Experiments
Issue number52
DOIs
StatePublished - Jun 2011

Fingerprint

Brain
Leukocytes
Flow cytometry
Isoflurane
Infection
Flow Cytometry
Theilovirus
Population
Syringes
Immunophenotyping
Ficoll
Grinding mills
Frontal Lobe
Myelin Sheath
Inbred C57BL Mouse
Viruses
Debris
Suspensions
Buffers
Hand

Keywords

  • Brain
  • Flow cytometry
  • Issue 52
  • Leukocytes
  • Mouse
  • Neuroimmunology
  • Neuroscience
  • Theiler's murine encephalomyelitis virus

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Isolation of brain-infiltrating leukocytes. / Lafrance-Corey, Reghann G.; Howe, Charles L.

In: Journal of Visualized Experiments, No. 52, e2747, 06.2011.

Research output: Contribution to journalArticle

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