Isolation of Brain-infiltrating Leukocytes

Summary

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

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.

Protocol

1. Intracranial virus injection:

The following technique has been modified and utilized extensively by our lab and colleagues. Briefly, intracranial injection of the Daniel's strain of Theiler's murine encephalomyelitis virus (TMEV) or sham-infection (1, 10) is performed on young mice (preferably 5-6 weeks of age) to elicit brain infiltrating leukocytes (BILs). Please note that results will differ between the Daniel's strain, the BeAN strain, and the GDVII strain. For the purpose of harvesting BILs, mice receive 2x10⁵ PFU of TMEV and are infected for 24 hours.

1. Attach the injection needle (27 gauge, ¼ in, Kendall), to a....

Discussion

We routinely use flow cytometry to determine both the quality of the brain infiltrating cell preparation, and to distinguish different populations of immune cells ^{2, 9}. At acute time-points, our BILs method yields high percentages of inflammatory monocytes within the CD45hi population as well as high percentages of macrophages in the CD45lo population. This indicates that a reproducible immune response within the brain can robustly be characterized by our method.

This technique is .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
TMEV	Howe Lab	N/A
Daniel’s strain	Invitrogen	21063-029
isoflurane	Novaplus	NDC 0409-3292-49
round-bottom Oak Ridge centrifuge tubes	Nalge Nunc international	3118-0030
RPMI 1640	Invitrogen	11875-093
Percoll	GE Healthcare	17-0891-02
10X PBS	Roche Group	11666789001
Ficoll-Paque Plus	GE Healthcare	17-1440-02
Trypan Blue 0.4% (w/v)	Mediatech, Inc.	25-900-CI
15 ml conical tubes	BD Biosciences	352097
7 mL glass Pyrex brand Tenbr–ck tissue grinder	Fisher Scientific	08-414-10B
40 μm cell strainer	BD Biosciences	352340
50 mL conical	BD Biosciences	352070
bovine serum albumin	Sigma-Aldrich	A9647
sodium azide	Sigma-Aldrich	S8032
CMF-PBS	Mediatech, Inc.	21-040-CV
FACS tubes	BD Biosciences	352054
fetal bovine serum	Sigma-Aldrich	F4135
2.4G2 hybridoma	ATCC	HB-197
Costar V-bottom plate	Corning	3894
Allegra X-22R centrifuge or equivalent	Beckman Coulter Inc.	N/A
96-well plate bucket and rotor	Beckman Coulter Inc.	S2096
Fixed-angle rotor	Beckman Coulter Inc.	F0360
paraformaldehyde	Sigma-Aldrich	P6148
BD FACS Calibur	BD Biosciences	N/A
FlowJo 7.5	Tree Star, Inc.	N/A
CD45	BD Biosciences	557235	clone: 30-F11
Gr1 (Ly6C/G)	BD Biosciences	553128	clone: RB6-8C5
CD11b	eBioscience	17-0112-83	clone: M1/70
F4/80	eBioscience	17-4801-82	clone: BM8