This work was supported by grants from the scholarship granted by the National Council of Science and Technology (CONACYT) (702361). The authors acknowledge the Ph.D. program in Biological Chemical Sciences of the National School of Biological Sciences of the National Polytechnic Institute.

The study was conducted in accordance with the official Mexican standard NOM-062-ZOO-1999 and the guide for the care and use of laboratory animals. Approval for the study was obtained from the Research, Ethics, and Biosafety Committees of the Mexico Children&#39;s Hospital (HIM/2023/006) and the Research and Bioethics Committee of the General Hospital of Mexico Eduardo Liceaga (DI/21/501/04/62). Three C57BL/6 mice aged 6 to 8 weeks were obtained from the Mexico Children&#39;s Hospital, where they were raised under isolated conditions in ventilated racks, in compliance with institutional animal care and use guidelines.
1. Preparation of materials and reagents
<ol>
	<li>Pre-warm Ca2+ and Mg2+ free PBS as well as Hank&#39;s balanced salt solution (HBSS) to 37 &#176;C.</li>
	<li>Supplement the HBSS solution with 100 &#181;g/mL of Liberase&#160;and 4 &#181;g/mL of DNase (HBSS-LD). Ensure that the solution maintains a physiological pH of 7.4. 
	NOTE: 1 mL of working solution per spinal cord will be necessary.</li>
	<li>Prepare an isotonic 90% solution for density gradient centrifugation (commercially available, see Table of Materials) and prewarm it to 37 &#176;C.</li>
	<li>Prepare the staining buffer by combining 5% heat-inactivated fetal bovine serum (FBS) with PBS, and store the mixture on ice.</li>
	<li>Pre-warm Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (1g/L) (DMEM) to 37 &#176;C.</li>
	<li>Supplement the DMEM medium with 10% FBS, 2 mM L-glutamine, 200 U/mL penicillin, 200 &#181;g/mL of streptomycin, and 500 pg/mL of amphotericin B (see Table of Materials) to prepare DMEM-S.</li>
</ol>
2. Dissection and preparation of the spinal cord
<ol>
	<li>Euthanize the mouse with an intraperitoneal overdose of pentobarbital at a dose of 150 mg/kg. Sterilize the mouse&#39;s thorax and back using 70% ethanol. 
	NOTE:&#160;Confirm euthanasia was successful before proceeding with the mouse dissection.</li>
	<li>Shave the thoracolumbar region and secure the extremities to the dissection board using adhesive tape.</li>
	<li>Using a scalpel, make a careful incision along the dorsal midline from the occipital to the sacral region (Figure 2).</li>
	<li>With the assistance of a stereoscopic surgical microscope, dissect in layers until reaching the lumbar spine and identifying the last costal arch. 
	NOTE: The 13 costal arches are articulated in the thoracic vertebrae; when dissected, they become visible superior to the L1 vertebra2.</li>
	<li>Determine the last cervical vertebra and the sacrum, then make a cut to separate the spine (Figure 2).</li>
	<li>Utilizing dissecting forceps, perform a dorsal laminectomy of the vertebrae to extract the spinal cord (Figure 3A-D). 
	NOTE: Except for the first two cervical vertebrae (atlas and axis), all movable vertebrae share a common morphological design in various regions (cervical, thoracic, or lumbar). Each typical movable vertebra features a cylindrical vertebral body at its anterior end. Attached to the back of the body is a bony arch known as the vertebral arch (neural arch), consisting of laminae and spinous processes. Between these structures lies the vertebral foramen15.</li>
	<li>Remove the nerves emerging through the foramina (dorsal and ventral roots) that constitute the peripheral nervous system and may contain infiltrated macrophages (Figure 3D). 
	NOTE: It is unnecessary to remove the pia mater or meninges as the density gradient medium effectively eliminates contaminating astrocytes. Additionally, retaining the meninges reduces purification time and enhances viability.</li>
</ol>
3. Tissue digestion and isolation of microglia
<ol>
	<li>Place the spinal cord on the dissecting board, and using Vannas scissors, cut the spinal cord into 2 mm pieces, yielding an average of 10 pieces.</li>
	<li>Transfer the sections of the spinal cord to a 2 mL flat-bottom microtube (Figure 3E,F).</li>
	<li>Add 1 mL of the HBSS-LD working solution (step 1.2). Incubate at 37 &#176;C for 25 min, vigorously vortexing every 5 min.</li>
	<li>Pass the contents of each digestion through a 40 &#181;m strainer, and then add 7 mL of HBSS with 2% FBS to neutralize the digestion.</li>
	<li>Crush the remaining tissue using a 1 mL syringe plunger. Centrifuge the suspension in a 50 mL conical tube for 5 min at 220 x g, 4 &#176;C.</li>
	<li>Discard the supernatant using a 1 mL micropipette, and resuspend the pellet in 2 mL of HBSS with 2% FBS in a 15 mL conical tube. Combine with 2 mL of 90% isotonic density gradient medium. Centrifuge for 25 min at 160 x g, 18 &#176;C. 
	NOTE: Utilize slow acceleration and avoid using the brake.</li>
	<li>Use a transfer pipette to retrieve the interface and transfer it to a 15 mL conical tube. Wash the cells with 10 mL of PBS and centrifuge for 5 min at 220 x g, 4 &#176;C.</li>
	<li>Resuspend the cells in DMEM-S (step 1.6) and keep them stored on ice.</li>
</ol>
4. Determining purity/viability
<ol>
	<li>For quantifying viable cells using a hemocytometer chamber, employ a 0.4% Trypan blue stain (see Table of Materials). 
	NOTE: On average, a single mouse provides 4-6 million cells.</li>
	<li>To quantify viable cells via a flow cytometer, employ commercially available amine-reactive fluorescent dye staining (see Table of Materials).
	<ol>
		<li>Transfer 1 million cells into a 5 mL polystyrene round-bottom (FACS) tube. Dilute the fluorescent dye with PBS at a ratio of 1:1000 (creating the viability working solution).</li>
		<li>Incubate the samples with the viability solution in the dark at 4 &#176;C for 30 min. Wash the cells with ice-cold PBS twice. Resuspend the cells in 400 &#181;L of staining buffer (step 1.4).</li>
		<li>Block the cells with 2.4G2 anti-FcRIII/I (see Table of Materials) in the ice-cold staining buffer for 15 min at 4 &#176;C.</li>
		<li>Formulate the antibody staining cocktail by adding the fluorescent-labeled monoclonal antibodies CD45-PerCP and CD11b-eFluor 450 to the staining buffer (at a dilution of 1:300) (see Table of Materials). 
		NOTE: While both techniques are available, choose the one that best aligns with the subsequent protocol.</li>
		<li>Incubate the samples with the antibody staining cocktail in the dark at 4 &#176;C for 30 min. Wash the cells with ice-cold staining buffer twice. Resuspend the cells in 400 &#181;L of staining buffer.</li>
		<li>Acquire 250,000 events on a flow cytometer, as outlined by the gating strategy in step 6 (Figure 4).</li>
	</ol>
	</li>
</ol>
5. Immunofluorescent staining
<ol>
	<li>Place a pretreated coverslip into a well of a 6-well plate. 
	NOTE: To treat coverslips, place them in a Petri dish with 500 &#181;L of L-polylysine (0.01 mg/mL, see Table of Materials) solution and incubate for 1 h. Afterward, wash them three times with distilled water and allow them to dry.</li>
	<li>Seed 200,000 cells onto the treated coverslip using 500 &#181;L of DMEM-S solution. Incubate for 24 h.</li>
	<li>Remove the coverslips and fix the cells using 3.7% paraformaldehyde (PFA) for 20 min at room temperature. Wash the coverslips three times with ice-cold PBS.</li>
	<li>Permeabilize the cells with 0.2% Triton X-100 in PBS for 15&#160;min at room temperature. Subsequently, wash the coverslips three times with cold PBS. Block the cells with 0.2% BSA in PBS for 1 h at room temperature.</li>
	<li>Dilute the fluorescence-labeled monoclonal antibodies (at a 1:300 dilution, see Table of Materials) in the blocking solution and incubate for 1 h at room temperature. Wash the coverslips three times with ice-cold PBS.</li>
	<li>Apply mounting medium containing DAPI onto the slide, and then seal it with a coverslip. 
	NOTE: The slides can be immediately analyzed using a fluorescence or confocal microscope, or they can be stored for up to 3 months at -20&#176;C.</li>
</ol>
6. Gating strategy for spinal cord microglia
<ol>
	<li>Generate a dot plot and establish a gate to isolate viable cells based on viability staining and an empty fluorescent channel16. Exclude non-viable cells.</li>
	<li>Produce another plot utilizing forward and side scatter parameters to eliminate debris16.</li>
	<li>Develop an additional dot plot and analyze the expression of CD11b and CD45 to differentiate microglia.</li>
</ol>

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

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	<li>Schr&#246;der, H., Schr&#246;der, , Moser, , Huggenberger, , 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=.">.</a> Neuroanatomy of the Mouse. , 59-78 (2020).</li><li>Sengul, G., 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=Cytoarchitecture+of+the+spinal+cord+of+the+postnatal+(P4)+mouse.">Cytoarchitecture of the spinal cord of the postnatal (P4) mouse.</a> Anat Rec. 295, 837-845 (2012).</li><li>Bab, I., 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=.">.</a> Microtomographic atlas of the mouse skeleton. VIII, 205 (2007).</li><li>Nayak, D., 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=Microglia+development+and+function.">Microglia development and function.</a> Annu Rev Immunol. 32, 367-402 (2014).</li><li>Martinez, F. O., 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=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front Biosci. 13, 453-461 (2008).</li><li>Masuda, 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=Microglia+heterogeneity+in+the+single-cell+era.">Microglia heterogeneity in the single-cell era.</a> Cell Rep. 30 (5), 1271-1281 (2020).</li><li>Prinz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+biology:+one+century+of+evolving+concepts.">Microglia biology: one century of evolving concepts.</a> Cell. 179 (2), 292-311 (2019).</li><li>de Haas, A. H., 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=Region-specific+expression+of+immunoregulatory+proteins+on+microglia+in+the+healthy+CNS.">Region-specific expression of immunoregulatory proteins on microglia in the healthy CNS.</a> Glia. 56 (8), 888-894 (2008).</li><li>Xuan, F. L., 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=Differences+of+microglia+in+the+brain+and+the+spinal+cord.">Differences of microglia in the brain and the spinal cord.</a> Front Cell Neurosci. 13, 504 (2019).</li><li>Paolicelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+states+and+nomenclature:+A+field+at+its+crossroads.">Microglia states and nomenclature: A field at its crossroads.</a> Neuron. 110 (21), 3458-3483 (2022).</li><li>Li, Q., 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=Spinal+IL-36&#947;/IL-36R+participates+in+the+maintenance+of+chronic+inflammatory+pain+through+astroglial+JNK+pathway.">Spinal IL-36&#947;/IL-36R participates in the maintenance of chronic inflammatory pain through astroglial JNK pathway.</a> Glia. 67 (3), 438-451 (2019).</li><li>Prinz, M., 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=Microglia+and+central+nervous+system-associated+macrophages-from+origin+to+disease+modulation.">Microglia and central nervous system-associated macrophages-from origin to disease modulation.</a> Annu Rev Immunol. 39, 251-277 (2021).</li><li>Yip, P. K., 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=Rapid+isolation+and+culture+of+primary+microglia+from+adult+mouse+spinal+cord.">Rapid isolation and culture of primary microglia from adult mouse spinal cord.</a> J Neurosci Methods. 183 (2), 223-237 (2009).</li><li>Akhmetzyanova, E. R., 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=Severity-+and+time-dependent+activation+of+microglia+in+spinal+cord+injury.">Severity- and time-dependent activation of microglia in spinal cord injury.</a> Int J Mo. 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The authors declare no conflicts of interest.

Numerous protocols have been developed for the study of microglia due to their significance in brain homeostasis. In these methods, microglia are typically sourced from the cerebral hemispheres of embryonic or neonatal rats and mice17. A limited number of studies have addressed the purification of microglia from the spinal cords of adult mice13,14. These techniques involve enzymatic digestion using collagenase and/or papain along with DNAs...

The defining characteristic of vertebrates is the vertebral column or spine, in which the notochord has been replaced by a sequence of segmented bones called vertebrae, divided by intervertebral discs. This succession of osseous material shapes the spinal canal, a cavity that encloses and protects the spinal cord1. In the genus Rodentia, the spine is usually formed by seven cervical vertebrae, thirteen thoracic vertebrae, six lumbar vertebrae, and a variable number of caudal vertebrae2,3. The length of the spinal cord is similar to that of the spine, and the terminal filum is a non-nervous structure that anchors the spinal cord to the sacrum. Additionally, nerve fibers exit through the intervertebral foramen1.
The development and proper function of the central nervous system in mammals critically depend on the activity of the nervous system&#39;s resident macrophages, called microglia4. Although microglia were initially described as brain resident phagocytes, recent research has attributed many dynamic functions to these cells5,6. Microglia&#39;s size ranges from 7 to 10 &#181;m in homeostasis; they are considered among the most versatile cells in the body and can adapt morphologically and functionally to their constantly changing environment7. These cells exhibit high heterogeneity during both the embryonic and adult stages8,9, while in the adult stage, they also display complex functional heterogeneity based on their spatiotemporal context10. The heterogeneity and multiple functions of microglia allow for differential gene expression and behavior in the spinal cord and brain. It has been shown that CD11b, CD45, CD86, and CCR9 expression is higher in the spinal cord compared to the brain8,9.
Multiple protocols exist for cerebral microglia isolation11,12; however, only a few exist for spinal cord microglia13,14. Optimizing a method for purifying microglia from the spinal cord facilitates the development of multiple studies focused on discovering microglia physiology. This protocol aims to describe a simple and highly reproducible extraction of the mouse spinal cord and the purification of microglia (Figure 1).

<table><tbody><tr><td>15 mL collection tubes</td><td>Corning, USA</td><td>430790</td><td></td></tr><tr><td>2 mL microtubes</td><td>Axygen, USA</td><td>MCT-200-G</td><td></td></tr><tr><td>2.4G2 anti-FcR</td><td>BioLegend, USA</td><td>101302</td><td></td></tr><tr><td>50 mL collection tubes</td><td>Corning, USA</td><td>430829</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b)</td><td>Gibco, USA</td><td>15240062</td><td></td></tr><tr><td>Antibody CD11b eFluor 450 anti-mouse</td><td>eBioscience, USA</td><td>48-0112</td><td></td></tr><tr><td>Antibody CD45 PerCP anti-mouse&amp;nbsp;&amp;nbsp;</td><td>BioLegend, USA</td><td>103130</td><td></td></tr><tr><td>Balanced salt solution (PBS) calcium- magnesium-free</td><td>Corning, USA</td><td>46-013-CM</td><td></td></tr><tr><td>Blue Cell Strainer 40 &amp;mu;m</td><td>Corning, USA</td><td>352340</td><td></td></tr><tr><td>Costar 6-well Clear Not Treated&amp;nbsp;</td><td>Corning, USA</td><td>CLS3736</td><td></td></tr><tr><td>Coverslips</td><td></td><td></td><td></td></tr><tr><td>Digital Heating Shaking Drybath&amp;nbsp;</td><td>Thermo Scientific Digital HS Drybath, USA</td><td>88870001</td><td></td></tr><tr><td>Dissecting forceps for microsurgery</td><td>FT by DUMONT</td><td></td><td></td></tr><tr><td>DNase</td><td>Roche, USA</td><td>4536282001</td><td></td></tr><tr><td>Dulbecco&amp;acute;s Modified Eagle&amp;acute;s Medium-high glucose (DMEM)&amp;nbsp;</td><td>Merck, USA</td><td>D6429</td><td></td></tr><tr><td>Electric shaver</td><td></td><td></td><td></td></tr><tr><td>FACS tube</td><td>Thermo, USA</td><td>352058</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>PAN Biotech, Alemania</td><td>P30-3306</td><td></td></tr><tr><td>Flow cytometer Cytoflex&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Merck, USA</td><td>H2387</td><td></td></tr><tr><td>L-glutamine</td><td>Corning, USA&amp;nbsp;</td><td>15393631</td><td></td></tr><tr><td>Liberase TM&amp;nbsp;</td><td>Roche, USA</td><td>5401119001</td><td></td></tr><tr><td>Neubauer chamber Counting Chambers</td><td>China</td><td>1103</td><td></td></tr><tr><td>Pentobarbital</td><td></td><td></td><td></td></tr><tr><td>Percoll&amp;nbsp;</td><td>Merck, USA</td><td>17089101</td><td>density gradient centrifugation&amp;nbsp;</td></tr><tr><td>Poly-L-lysine solution&amp;nbsp;</td><td>Merck, USA</td><td>P8920</td><td></td></tr><tr><td>Scalpel No. 25&amp;nbsp;</td><td>HERGOM, Mexico</td><td>H23</td><td></td></tr><tr><td>Snaplock Microcentrifuge Tubes 2 mL</td><td>Axygen, USA</td><td>10011-680</td><td></td></tr><tr><td>Stereoscopic microscope</td><td>Velab, Mexico</td><td>HG927831</td><td></td></tr><tr><td>Straight surgical scissors (10 cm)</td><td>HERGOM, Mexico</td><td></td><td></td></tr><tr><td>Straight Vannas scissors</td><td>HERGOM, Mexico</td><td></td><td></td></tr><tr><td>Triton X100</td><td>Merck, USA</td><td>X100</td><td></td></tr><tr><td>Trypan blue Stain 0.4%&amp;nbsp;</td><td>Merck, USA</td><td>15250-061</td><td></td></tr><tr><td>Vortex mixer</td><td>DLAB, China</td><td>8031102000</td><td></td></tr><tr><td>Zombie Aqua Fixable Viability Kit</td><td>BioLegend, USA</td><td>423102</td><td>amine-reactive fluorescent dye staining&amp;nbsp;</td></tr></tbody></table>

rapid and efficient enrichment of mouse spinal cord microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Utilizing mouse spinal cord tissue, enzymatic digestion was performed using a mixture highly enriched with collagenase and thermolysin. The resulting digested tissue underwent passage through a 40 &#181;m filter to eliminate undigested material. The collected cells were enriched through a Percoll density gradient, with 90% in the lower portion and 45% in the upper portion. The microglia-enriched cells within the interface were then stained with CD45 and CD11b antibodies and subjected to flow cytometric analysis (<strong ...

Watch this Scientific Journal Video about Microglia Research on Spinal Cord Heterogeneity and Purification at JoVE.com

Author Spotlight: Microglia Research on Spinal Cord Heterogeneity and Purification 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

rapid-and-efficient-enrichment-of-mouse-spinal-cord-microglia

Research

JoVE Journal

Neuroscience

1.5K Views.  Mexico Children's Hospital. Ons onderzoek richt zich op de heterogeniteit van microglia, die zowel pro- als anti-inflammatoir gedrag kunnen vertonen in verschillende pathologieën en regio's zoals de hersenen en het ruggenmerg. We streven ernaar om een beter begrip te krijgen van het gedrag van microglia in het ruggenmerg in verschillende scenario's. De heterogeniteit van microglia heeft het moeilijk gemaakt om ze te classificeren.Dit jaar is een nieuwe en brede classificatie voorgesteld die verder gaat dan allee...

Video: Auteur in de schijnwerpers: Microglia-onderzoek naar heterogeniteit en zuivering van het ruggenmerg 

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 ...

Immunology and Infection

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

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 ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

Source: Maguire, A. D., et al. <a href="https://app.jove.com/v/64603">Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.</a> J. Vis. Exp. (2023)
This video demonstrates a protocol for enriching neuronal cultures from dorsal root ganglia by immunopanning, thereby improving their purity and utility for neurological research.

1. Experimental method

	Day 1: preparation of dishes
	
		In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the ...

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

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