This work was supported by NIEHS R01ES014470 (KMZ).

&#160;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.&#160;Preparation of instruments, Culture Media, and Dishes
<ol>
	<li>Prepare 10 ml/pup Microglia Complete Media (MCM; 1x Minimal Essential Medium Earle&#39;s [MEM] supplemented with: 1 mM L-glutamine, 1 mM sodium pyruvate, 0.6% v/v D-(+)-glucose, 100 &#956;g/ml&#160;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 &#176;C, 5% CO2) to equilibrate for 1 hr. It is essential to equilibrate this media in flask(s) before starting the dissection.</li>
	<li>Prepare, aliquot, and warm in 37&#160;&#176;C water bath&#160;MCM for dissection&#160;(6 ml/pup) and in a separate conical tube MCM for cell resuspension (2 ml/pup).&#160;Prepare, aliquot, and&#160;chill on ice&#160;Mincing Media (MM; HBSS supplemented with: 100 &#956;g/ml&#160;P/S, 0.01 M HEPES; 3 ml/pup), and Dissecting Media (DM; MEM supplemented with 100 &#956;g/ml&#160;P/S; 2 ml/pup).</li>
	<li>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.</li>
	<li>Wipe down the horizontal-airflow workstation and dissection microscope with 70% ethanol.</li>
	<li>Dispense DM into 2 sterile, 100&#160;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&#160;mm Petri dish for mincing brain tissue (1 ml; 1 dish/pup).</li>
</ol>
2.&#160;Brain Tissue Dissection
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3.&#160;Brain Tissue Mincing
<ol>
	<li>Transfer cortices to previously prepared mincing dish; continue to mincing step using sterile technique in Class II Biological Safety Cabinet.</li>
	<li>Mince brain tissue using scissor #3; transfer minced tissue to a 15&#160;ml&#160;conical tube; rinse scissors and mincing dish with 1 ml&#160;of MM each, collect and dispense rinses into the 15 ml&#160;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&#160;as MM does not contain essential nutrients.</li>
	<li>Carefully remove and discard 2 ml&#160;of supernatant with generic 1,000 &#956;l&#160;micropipettor with a standard pipette tip, making certain not to disrupt the settled minced-tissue.</li>
	<li>Add 3 ml&#160;of MCM to the minced-tissue; triturate the tissue with full force using a generic 1,000 &#956;l&#160;micropipettor with a standard pipette tip. No discernable solid tissue should remain when trituration is complete. Add another 3 ml&#160;of MCM with the same pipette tip, collecting residual triturated sample from the pipette tip.</li>
</ol>
4.&#160;Growing Cortical Cells
<ol>
	<li>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 &#176;C, 5% CO2). Allow the cells to incubate for 24 hr.</li>
	<li>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 &#176;C); cap and place back into the incubator, horizontally.</li>
	<li>Check cells daily for contamination and to monitor growth.</li>
	<li>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.</li>
</ol>
5.&#160;Isolation of Microglia from Mixed Glial Culture
<ol>
	<li>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.</li>
	<li>Seal T-75 flask caps with Parafilm and rotate the mixed glial cultures (200 rpm; 37 &#176;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.</li>
	<li>Collect supernatant media containing microglia into a sterile conical centrifuge tube; pellet microglia (215 x g; 5 min; swinging bucket rotor).</li>
	<li>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 &#956;g/ml&#160;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 &#956;l/well; MGM). Using this procedure the average microglia yield is 7.5 x 105 cells/pup cortex.</li>
	<li>24-hr&#160;post-plating, remove all of the media and refeed cells with fresh MGM (37 &#176;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.</li>
</ol>
6.&#160;Example Treatments
<ol>
	<li>Expose cells to 10 ng/ml&#160;of Pam3CSK4&#160;(Pam), 10 ng/ml&#160;lipopolysaccharide (LPS) or vehicle control; incubate for 2 or 24 hr&#160;in humidified incubator (37 &#176;C, 5% CO2).</li>
</ol>
7.&#160;Analyzing Microglia for Functionality Ex vivo via Immunofluorescent Imaging and Immunocytochemistry
<ol>
	<li>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 &#176;C until ready to use. Analyze TNF-&#945; concentration in cell culture supernatant via commercially available mouse TNF-&#945; ELISA kit.</li>
	<li>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&#160;with gentle shaking on an orbital shaker.
	<ol>
		<li>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&#160;at 4 &#176;C with rabbit anti-NF-&#954;B 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).</li>
		<li>Counter stain cells with 4&#39;,6-Diamidino-2-Phenylindole/PBS (DAPI; 0.1 &#956;g/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.</li>
	</ol>
	</li>
	<li>Mount cover slips using aqueous mounting solution and visualize microglia using fluorescent microscopy.</li>
</ol>

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Res. 16, 238-254 (2009).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus:immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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...

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&#160;and vary in cell density and morphology in different cytoarchitectural regions within the adult CNS1-5. 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 &#34;sampling and surveying&#34;6,7.
Microglia are highly and differentially responsive to environmental and pathophysiological alterations in the brain, switching from a surveillant to an effector state&#8212;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 carbohydrates8-11. 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 damage12-16. 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-&#954;B subunit (p65) to the cell nucleus, and upregulate the production and secretion of proinflammatory cytokines, such as tumor necrosis factor-&#945; (TNF-&#945;), interleukin-1 &#946; (IL-1&#946;), 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&#39;s and Alzheimer&#39;s disease25-29.
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 tissue30,31. 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 cells30,33,34. 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 method24,35. We utilize the former mouse strain for easy visualization of microglia, as these mice express GFP under the control of the endogenous Cx3Cr1 locus -&#160;a monocyte-specific promoter36-38. 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-&#945; when challenged with bacterial lipopolysaccharide (LPS) or Pam3CSK4, TLR4 and TLR1/2 agonists, respectively.

<table><tbody><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &amp;deg;C; shelf life: 3-6 months. Used to make MCM and MGM.</td></tr><tr><td>Sodium pyruvate 100 mM (100x)</td><td>Hyclone</td><td>SH30239.01</td><td>Store at 4 &amp;deg;C. Used to make MCM and MGM.</td></tr><tr><td>Penicillin/Streptomycin 10,000 units/ml (100x)</td><td>Gibco</td><td>15140-122</td><td>Store at -20 &amp;deg;C. Used to make MCM, MGM, MM, and DM.</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco</td><td>25030-081</td><td>Store at -20 &amp;deg;C. Used to make MCM and MGM.</td></tr><tr><td>Fetal Bovine Serum (Defined)</td><td>Hyclone</td><td>SH30070.03</td><td>Filter sterilize; store at -20 &amp;deg;C. Used to make MCM and MGM.</td></tr><tr><td>Minimum Essential Medium Earle&#039;s (MEM)</td><td>Cellgro</td><td>15-010-CV</td><td>Without L-glutamine. Contains Earle&#039;s salts. Used to make MCM, MGM, and DM.</td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050</td><td>Filter sterilize; store at -20 &amp;deg;C. Used to make MCM and MGM.</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Cellgro</td><td>21-021-CV</td><td>Without calcium and magnesium. Store at 4 &amp;deg;C. Used to make MM.</td></tr><tr><td>HEPES 1 M</td><td>Gibco</td><td>15630-031</td><td>Store at 4 &amp;deg;C. Used to make MM.</td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>4&#039;,6-Diamidino-2-phenylindole, dilactate (DAPI)</td><td>Invitrogen</td><td>D3571</td><td>Used to stain cell nucleus.</td></tr><tr><td>Rabbit anti-Iba1</td><td>Wako</td><td>019-19741</td><td>Used at 1/750 dilution for ICC staining of Iba1.&amp;nbsp;</td></tr><tr><td>Rabbit anti-NF&amp;kappa;B (p65)</td><td>Abcam</td><td>7970</td><td>Used at 1/1,250 dilution for ICC staining for p65.</td></tr><tr><td>Alexa Fluor 594 Goat anti-Rabbit IgG (H+L)</td><td>Invitrogen</td><td>A11012</td><td>Used at 1/1,000 dilution for visualization of antigen:antibody complex in ICC.</td></tr><tr><td>10 ml Disposable Serological Pipet</td><td>Fisher Scientific</td><td>13-678-11E</td><td></td></tr><tr><td>50 ml Disposable Centrifuge Tube</td><td>Fisher Scientific</td><td>05-539-8</td><td></td></tr><tr><td>15 ml Disposible Centrifuge Tube</td><td>Fisher Scientific</td><td>05-539-12</td><td></td></tr><tr><td>Sterile Polystyrene Petri Dish</td><td>Fisher Scientific</td><td>875713</td><td>100 mm x 15 mm</td></tr><tr><td>Scissor: Straight Metzembaum (scissor #1)</td><td>Roboz Surgical</td><td>RS-6010</td><td>1; 5 in; used for removing head</td></tr><tr><td>Scissor: Vannas&lt;br/&gt; (scissor #2)</td><td>Fine Science Tools</td><td>15000-08</td><td>1; nonangled; 2.5 mm cutting edge; used to open scalp</td></tr><tr><td>Scissor: Student Vannas (scissor #3)</td><td>Fine Science Tools</td><td>91501-09</td><td>1; curved; used to mince brain tissue</td></tr><tr><td>Forcep: Dumont #7&lt;br/&gt; (forcep #1)</td><td>Fine Science Tools</td><td>91197-00</td><td>2; used to secure nose and remove cortices&amp;nbsp;</td></tr><tr><td>Forcep: Dumont #2&lt;br/&gt; (forcep #2)</td><td>Fine Science Tools</td><td>11223-20</td><td>1; used to remove scalp</td></tr><tr><td>Forcep: Dumont #3&lt;br/&gt; (forcep #3)</td><td>Fine Science Tools</td><td>11231-30</td><td>1; used to remove skull</td></tr><tr><td>Forcep: Dumont #5a&lt;br/&gt; (forcep #4)</td><td>Fine Science Tools</td><td>11253-21</td><td>1; used to remove meninges</td></tr><tr><td>&lt;strong&gt;Table of Specific Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zoom Stereo Dissection Microscope&amp;nbsp;</td><td>Olympus</td><td>SZ4060</td><td>Microscope is placed inside Laminar-Horizontal Flow Cabinet</td></tr><tr><td>Laminar-Horizontal Flow Cabinet</td><td>Nuaire</td><td>NU-201-330</td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>Labconco</td><td>3440001</td><td>Class II</td></tr><tr><td>Water-Jacketed CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>VWR</td><td>97025-836</td><td>Set to 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Swing-out buckets</td><td>Fisher Scientific</td><td>75006441</td><td>To be used with Swing-out rotor</td></tr><tr><td>Swing-out Rotor</td><td>Fisher Scientific</td><td>75006445</td><td>Max Radius: 19.2 (cm)</td></tr><tr><td>Sorvall Legend RT+ Centrifuge&lt;br/&gt; (clinical centrifuge)</td><td>Fisher Scientific</td><td>75-004-377</td><td>With swing-out rotor</td></tr><tr><td>AccuSpin Micro 17 microcentrifuge&lt;br/&gt; (tabletop microcentrifuge)</td><td>Fisher Scientific</td><td>S98645</td><td>With microliter rotor (24 x 1.5/2.0 ml; Cat #: 75003524)&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

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-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), 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.

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...

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

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).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

isolation-cortical-microglia-with-preserved-immunophenotype

Universidad de Cartagena UDEC

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Immunology and Infection

15.7K Views.  Georgetown University Medical Center. 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).

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

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

Izolacja mikrogleju z kory mózgowej dorosłej myszy

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

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Neuronal Culture Techniques

Primary Neuronal Culture

Magnetic Isolation of Microglia from Mouse Pup Brains

Source: Bokobza, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62964">Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures.</a> J. Vis. Exp. (2022)
In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell population.

Izolacja magnetyczna mikrogleju z mózgów młodych myszy

Source: Bokobza, C. et al., Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (2022)
In this video, we ...