Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Summary

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam₃CSK₄ (Pam).

Abstract

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-κB (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-α (TNF-α), upon lipopolysaccharide (LPS) and Pam₃CSK₄ (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

Introduction

Microglial cells, the surveillance macrophages of the CNS parenchyma, comprise approximately 12% of the total cell population of the adult mammalian brain. Microglia are derived from yolk sac myeloid precursor cells and vary in cell density and morphology in different cytoarchitectural regions within the adult CNS^1-5. In a healthy adult brain, microglia are small, ramified or polar cells with fine, dynamic processes. In contrast to peripheral macrophage morphology, microglia demonstrate a quiescent, low-profile phenotype in healthy brains that may appear as cellular inactivity; however, in vivo imaging studies demonstrate that microglial proce....

Protocol

 Prior to starting this protocol, collect neonatal mice at postnatal days 1-3 (P1-3) in a sterile container nested with original cage bedding for protection and warmth. It is important to work quickly and efficiently through this protocol to optimize microglial yield. Please see Table 1 for complete reagent list.

1. Preparation of instruments, Culture Media, and Dishes

1. Prepare 10 ml/pup Microglia Complete Media (MCM; 1x Minimal Essential Medium Earle's [MEM] supplemented with: 1 mM L-glutamine, 1 mM sodium pyruvate, 0.6% v/v D-(+)-glucose, 100 μg/ml Penicillin/Streptomycin (P/S), 4% v/v F....

Results

Retaining the immunophenotype and functionality of microglia ex vivo during isolation is critical to be able to utilize these cells as an investigatory model for microglial biology. In order to demonstrate the successful preservation of microglia immunofunctionality using the present method, we isolated cortical microglia from P3 neonates (CX3CR1-GFP^+/-and C57BL/6) and treated cultures with either LPS or Pam.

As illustrated in Figure 1A, isolation of microg.......

Discussion

The present procedure offers an effective method for the isolation of cortical microglia from neonatal mice. This procedure has a two-fold benefit of 1) preserving microglia immunophenotype and functionality, as determined by fluorescent imaging, immunocytochemistry, and ELISA; and, 2) allowing microglia to mature in the presence of other glial cells (astrocytes and oligodentrocytes) prior to isolation, which may facilitate important cell-cell interactions during the glial culture maturation period. Importantly, isolated.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glucose	Sigma	G8270	Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 °C; shelf life: 3-6 months. Used to make MCM and MGM.
Sodium pyruvate 100 mM (100x)	Hyclone	SH30239.01	Store at 4 °C. Used to make MCM and MGM.
Penicillin/Streptomycin 10,000 units/ml (100x)	Gibco	15140-122	Store at -20 °C. Used to make MCM, MGM, MM, and DM.
L-Glutamine 200 mM (100x)	Gibco	25030-081	Store at -20 °C. Used to make MCM and MGM.
Fetal Bovine Serum (Defined)	Hyclone	SH30070.03	Filter sterilize; store at -20 °C. Used to make MCM and MGM.
Minimum Essential Medium Earle's (MEM)	Cellgro	15-010-CV	Without L-glutamine. Contains Earle's salts. Used to make MCM, MGM, and DM.
Horse Serum	Gibco	16050	Filter sterilize; store at -20 °C. Used to make MCM and MGM.
Hanks' Balanced Salt Solution (HBSS)	Cellgro	21-021-CV	Without calcium and magnesium. Store at 4 °C. Used to make MM.
HEPES 1 M	Gibco	15630-031	Store at 4 °C. Used to make MM.
T-75 Flask	Corning	430641
4',6-Diamidino-2-phenylindole, dilactate (DAPI)	Invitrogen	D3571	Used to stain cell nucleus.
Rabbit anti-Iba1	Wako	019-19741	Used at 1/750 dilution for ICC staining of Iba1.
Rabbit anti-NFκB (p65)	Abcam	7970	Used at 1/1,250 dilution for ICC staining for p65.
Alexa Fluor 594 Goat anti-Rabbit IgG (H+L)	Invitrogen	A11012	Used at 1/1,000 dilution for visualization of antigen:antibody complex in ICC.
10 ml Disposable Serological Pipet	Fisher Scientific	13-678-11E
50 ml Disposable Centrifuge Tube	Fisher Scientific	05-539-8
15 ml Disposible Centrifuge Tube	Fisher Scientific	05-539-12
Sterile Polystyrene Petri Dish	Fisher Scientific	875713	100 mm x 15 mm
Scissor: Straight Metzembaum (scissor #1)	Roboz Surgical	RS-6010	1; 5 in; used for removing head
Scissor: Vannas (scissor #2)	Fine Science Tools	15000-08	1; nonangled; 2.5 mm cutting edge; used to open scalp
Scissor: Student Vannas (scissor #3)	Fine Science Tools	91501-09	1; curved; used to mince brain tissue
Forcep: Dumont #7 (forcep #1)	Fine Science Tools	91197-00	2; used to secure nose and remove cortices
Forcep: Dumont #2 (forcep #2)	Fine Science Tools	11223-20	1; used to remove scalp
Forcep: Dumont #3 (forcep #3)	Fine Science Tools	11231-30	1; used to remove skull
Forcep: Dumont #5a (forcep #4)	Fine Science Tools	11253-21	1; used to remove meninges
Table of Specific Equipment
Zoom Stereo Dissection Microscope	Olympus	SZ4060	Microscope is placed inside Laminar-Horizontal Flow Cabinet
Laminar-Horizontal Flow Cabinet	Nuaire	NU-201-330
Biological Safety Cabinet	Labconco	3440001	Class II
Water-Jacketed CO2 Incubator	VWR	97025-836	Set to 37 °C, 5% CO2
Swing-out buckets	Fisher Scientific	75006441	To be used with Swing-out rotor
Swing-out Rotor	Fisher Scientific	75006445	Max Radius: 19.2 (cm)
Sorvall Legend RT+ Centrifuge (clinical centrifuge)	Fisher Scientific	75-004-377	With swing-out rotor
AccuSpin Micro 17 microcentrifuge (tabletop microcentrifuge)	Fisher Scientific	S98645	With microliter rotor (24 x 1.5/2.0 ml; Cat #: 75003524)