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 processes dynamically extend and retract to monitor their microenvironment in a manner reminiscent of "sampling and surveying"^6,7.

Microglia are highly and differentially responsive to environmental and pathophysiological alterations in the brain, switching from a surveillant to an effector state—commonly regarded as their resting and activated states, respectively. This switch in activation can be mediated by engagement of membrane-bound pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), which respond to pathogen-associated molecular patterns (PAMPs), namely bacterial- and viral-derived lipoproteins, nucleic acids, and carbohydrates^8-11. In addition to PAMPs, PRRs have also been shown to induce microglial activation against sterile, nonpathogenic molecules known as danger/damage-associated molecular patterns (DAMPs), which represent a perturbation in CNS homeostasis, such as cellular damage^12-16. Once engaged, PRRs initiate an intracellular signaling cascade that results in changes in microglial cell morphology and gene expression; specifically, activated microglia adapt an amoeboid-like phenotype, translocate the p65 NF-κB subunit (p65) to the cell nucleus, and upregulate the production and secretion of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β), along with reactive oxygen species (ROS)^16-24. Although integral in the innate immune response of the CNS, these secreted molecules have also been found to increase neuronal oxidative stress, thereby inducing and exacerbating neurodegeneration in diseased states such as Parkinson's and Alzheimer's disease^25-29.

However, the mechanisms of microglial activation in pathological states are not completely understood. Therefore, isolation of microglia is a powerful investigative tool into these biological processes, as many in vivo features of microglial activation can be recapitulated in culture. Several methods are available for isolating microglia, including isolation via a Percoll gradient following enzymatic digestion of CNS tissue^30,31. However, enzymatic digestion can alter the immunophenotype of the cells by reducing cell-surface antigen expression³², and results in lower cell yield per animal than the method described herein. Specifically, we report an average microglia yield per pup cortex of 7.5 x 10⁵ cells, while previously reported isolation methods from whole CNS via a Percoll gradient yield 3-5 x 10⁵ cells^30,33,34. The present procedure circumvents the use of digestion enzymes by isolating microglia based on their low-adherence properties, thereby preserving the microglial immunophenotype and functionality.

In the present study, we describe the isolation of microglial cells from mixed glial cultures derived from neonatal heterozygous CX3CR1-GFP (CX3CR1-GFP^+/-), and C57BL/6 murine cortices via mechanical agitation on a rotary shaker, an extension of previously published method^24,35. We utilize the former mouse strain for easy visualization of microglia, as these mice express GFP under the control of the endogenous Cx3Cr1 locus - a monocyte-specific promoter^36-38. This method produces highly pure microglial cultures with a preserved immunophenotype ex vivo as demonstrated by morphological changes, nuclear translocation of p65, and secretion of TNF-α when challenged with bacterial lipopolysaccharide (LPS) or Pam₃CSK₄, TLR4 and TLR1/2 agonists, respectively.

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 Fetal Bovine Serum (FBS), 6% v/v Horse Serum), and add to each T-75 flask (1 flask/pup); cap and place horizontally into an incubator (37 °C, 5% CO₂) to equilibrate for 1 hr. It is essential to equilibrate this media in flask(s) before starting the dissection.
2. Prepare, aliquot, and warm in 37 °C water bath MCM for dissection (6 ml/pup) and in a separate conical tube MCM for cell resuspension (2 ml/pup). Prepare, aliquot, and chill on ice Mincing Media (MM; HBSS supplemented with: 100 μg/ml P/S, 0.01 M HEPES; 3 ml/pup), and Dissecting Media (DM; MEM supplemented with 100 μg/ml P/S; 2 ml/pup).
3. Sterilize the following surgical tools by submersion in 70% ethanol for 10 min: scissor #1 (1; to decapitate), forcep #1 (2; to secure nose and remove cortices), scissor #2 (1; nonangled; to open scalp), forcep #2 (1; to remove scalp), forcep #3 (1; to remove skull), forcep #4 (2; to remove meninges), scissor #3 (1; to mince brain tissue). Allow to air dry on a sterile pad.
4. Wipe down the horizontal-airflow workstation and dissection microscope with 70% ethanol.
5. Dispense DM into 2 sterile, 100 mm Petri dishes for capturing head (3 ml; 1 dish/3 pups) and dissection (1 ml; 1 dish/pup; chilled on ice prior to dissection). Dispense MM into 1 sterile, 100 mm Petri dish for mincing brain tissue (1 ml; 1 dish/pup).

2. Brain Tissue Dissection

1. Prior to euthanasia, gently sanitize neck and head of pup with 70% ethanol using a lab tissue. With scissor #1, quickly euthanize pup by removing the head; capture head in previously prepared Petri dish. Euthanize and complete the dissection of one pup before proceeding to the next animal.
2. Transfer the head to the chilled dissection dish; under dissection microscope secure the head by pinching the nose using forcep #1 (alternatively, the head can be secured by piercing the forceps through the eye socket); open scalp using scissor #2 by creating a Y-cut: begin from base of head and at an angle toward the eyes. Use forcep #3 to gently remove scalp by peeling laterally, exposing the skull.
3. Using forcep #3, gently remove connective tissue at base of head. When base of skull is exposed, carefully open skull by threading one arm of forcep in between the brain and skull (following the interhemispheric fissure); grip; gently pull the skull upwards to tear open.
4. Disrupt connective tissue under ventral side of brain using forcep #1, and subsequently remove brain using the same tool. Lift the brain out of skull by using an upward force under the ventral side of the brain.
5. Keep the brain in its anatomical position: dorsal side facing upwards to visualize the cortices under the dissection microscope; secure the brain in place by inserting tips of forceps in the intersection of the midbrain and the cortex; completely remove cortical meninges with forcep #4, then proceed to remove the ventral meninges. Once the meninges have been completely removed, separate the cortices from the remainder of the brain using forcep #4. It is essential to completely remove meningeal tissue to maximize microglia purity and yield.

3. Brain Tissue Mincing

1. Transfer cortices to previously prepared mincing dish; continue to mincing step using sterile technique in Class II Biological Safety Cabinet.
2. Mince brain tissue using scissor #3; transfer minced tissue to a 15 ml conical tube; rinse scissors and mincing dish with 1 ml of MM each, collect and dispense rinses into the 15 ml conical tube containing the minced tissue. This rinse step will maximize the yield of microglia by ensuring that all residual tissue is transferred to conical tube. Leave tissue suspension undisturbed for 1-2 min, allowing minced tissue to settle at bottom of tube. Do not exceed 2 min as MM does not contain essential nutrients.
3. Carefully remove and discard 2 ml of supernatant with generic 1,000 μl micropipettor with a standard pipette tip, making certain not to disrupt the settled minced-tissue.
4. Add 3 ml of MCM to the minced-tissue; triturate the tissue with full force using a generic 1,000 μl micropipettor with a standard pipette tip. No discernable solid tissue should remain when trituration is complete. Add another 3 ml of MCM with the same pipette tip, collecting residual triturated sample from the pipette tip.

4. Growing Cortical Cells

1. Centrifuge the minced tissue to pellet cells (215 x g; 5 min; RT; clinical centrifuge with swinging bucket rotor). Return pelleted cells to the biological safety cabinet, remove and discard the supernatant; gently resuspend the cell pellet (2 ml/tube; MCM); add the cell resuspension to the previously prepared T-75 flask; cap the flask and place it horizontally into a humidified, standard tissue culture incubator (37 °C, 5% CO₂). Allow the cells to incubate for 24 hr.
2. After 24 hr, gently tap flask against palm of hand to disrupt tissue debris. Check cells before and after tapping to ensure complete removal of debris. Set flask vertical; remove and discard media; add fresh MCM to the bottom of the flask (12 ml; 37 °C); cap and place back into the incubator, horizontally.
3. Check cells daily for contamination and to monitor growth.
4. At approximately 17-21 days in vitro (DIV), microglial cells will be ready for harvest from the mixed glial culture. To determine if cells are ready, examine cells under an inverted phase-contrast microscope. Microglia are ready for harvest when at ~40% confluency. They appear as small, bright, spherical cells, growing as a monolayer on top of other glia.

5. Isolation of Microglia from Mixed Glial Culture

1. To isolate microglia, vigorously tap flask against laboratory bench. This agitation helps separate microglia from other glial cells due to the low-adherence properties of microglia.
2. Seal T-75 flask caps with Parafilm and rotate the mixed glial cultures (200 rpm; 37 °C; 5 hr) using a temperature-controlled, nonhumidified rotary shaker. Do not allow cells to shake for longer than 5 hr. Visually inspect that microglia have lifted off the flask surface after 5 hr, using an inverted phase-contrast microscope. Microglia are bright, spherical, free-floating cells. There will be a residual layer of astrocytes and oligodendrocytes adhering to the bottom of the flask.
3. Collect supernatant media containing microglia into a sterile conical centrifuge tube; pellet microglia (215 x g; 5 min; swinging bucket rotor).
4. Resuspend the microglial pellet in warm Microglial Growth Medium (MGM; MEM supplemented with: 1 mM sodium pyruvate, 0.6% v/vglucose, 1 mM L-glutamine, 100 μg/ml P/S, 5% v/v FBS; ~2 ml/flask), determine the cell concentration using a hemocytometer, and plate 1 x 10⁵ cells/well in a 24-well plate format on plastic or sterile glass cover slips (500 μl/well; MGM). Using this procedure the average microglia yield is 7.5 x 10⁵ cells/pup cortex.
5. 24-hr post-plating, remove all of the media and refeed cells with fresh MGM (37 °C) to remove floating cells and debris. This step is critical to cell survival and to maintain quiescent microglia. Cells may be utilized for experimentation immediately after refeeding.

6. Example Treatments

1. Expose cells to 10 ng/ml of Pam₃CSK₄ (Pam), 10 ng/ml lipopolysaccharide (LPS) or vehicle control; incubate for 2 or 24 hr in humidified incubator (37 °C, 5% CO₂).

7. Analyzing Microglia for Functionality Ex vivo via Immunofluorescent Imaging and Immunocytochemistry

1. After treatment, harvest cell culture media in microfuge tubes (1.5 ml) and centrifuge using a tabletop microcentrifuge (2 min; 900 x g) to clear media. Transfer the supernatant to a clean microfuge tube; assay immediately, or store at -20 °C until ready to use. Analyze TNF-α concentration in cell culture supernatant via commercially available mouse TNF-α ELISA kit.
2. Wash cells with phosphate buffered saline (PBS), and fix cells with 4% (w/v) paraformaldehyde/4% (w/v) sucrose/PBS pH 7.4 (4% PFA; 15 min; RT). Following fixation, wash cells with PBS for 5 min with gentle shaking on an orbital shaker.
   1. Immunofluorescence/Immunocytochemistry. After fixation and PBS wash, process microglia for immunocytochemistry. All steps carried out with gentle shaking. Permeabilize cells in PBS/0.1% triton X-100; block cells in PBS/10% normal goat serum for one hour. Incubate cells O/N at 4 °C with rabbit anti-NF-κB antibody (p65 subunit; 1/1,250) or rabbit anti-Iba-1 antibody (1/750) in blocking buffer. Visualize antigen:antibody complex following incubation with fluorescently conjugated goat anti-rabbit IgG secondary antibody (1 hr; 1/1,000). Remove unbound secondary antibody by washing with PBS/0.1% triton X-100 (3x 5 min).
   2. Counter stain cells with 4',6-Diamidino-2-Phenylindole/PBS (DAPI; 0.1 μg/ml) to visualize the cell nucleus. All microglia derived from CX3CR1-GFP^+/- mice express GFP. Utilize a 350 nm wavelength filter to visualize DAPI; a 488 nm wavelength filter to visualize GFP; and a 594 nm wavelength filter to visualize p65 and Iba-1 immunocomplexes.
3. Mount cover slips using aqueous mounting solution and visualize microglia using fluorescent microscopy.

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)