We thank Castle Raley at the George Washington University Genomics Core for RNA analyses and Q2 Lab Solutions for RNA sequencing analyses. This work was supported by the National Institute of Neurological Disorders and Stroke [grant number F31NS117085] and the Vivian Gill Research Endowment to Dr. Robert H. Miller. Figure 1 was created with BioRender.com.

All animal care and experimental procedures were conducted following the approval of the Animal Care and Use Committee at The George Washington University School of Medicine and Health Sciences (Washington, D.C., USA; IACUC#2021-004). The study utilized male and female C57BL/6J wild-type (WT) mice aged 10 weeks to 5 months, which were sourced from a commercial supplier (see Table of Materials) and housed at The George Washington University. An overview of the protocol workflow is presented in Figure 1.
1. Preparation of the spinal cord
<ol>
	<li>Anesthetize the animals using Avertin (12.5 mg/mL of 2,2,2-Tribromoethanol and 2.5% 2-methyl-2-butanol in sterile water) (see Table of Materials). Confirm the absence of a response to toe and tail pinches in the mice.</li>
	<li>Perform cardiac perfusion with cold 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) to remove peripheral blood mononuclear cells.
	<ol>
		<li>Place the animal on a shallow tray and make a lateral incision to expose the pleural cavity. Insert a 23 G perfusion needle connected to a peristaltic pump (see Table of Materials) into the left ventricle and activate the pump. Once cold DPBS flows through the heart, create a small incision in the right atrium. 
		&#8203;NOTE: A faster perfusion rate is preferred in this step to enhance cell viability. Blood should be flushed out in under 1 min. Clearing of the liver indicates successful perfusion.</li>
	</ol>
	</li>
	<li>Decapitate the animal using large scissors and remove the spinal column14. Submerge it in cold 1x DPBS with Ca2+/Mg2+/0.2% glucose in a Petri dish on ice for tissue rinsing.</li>
	<li>Starting now, ensure that all steps are conducted in a sterile environment.&#160;Use hydraulic extrusion14 to isolate the entire spinal cord. Employ an 18 G needle and a 10 mL syringe filled with cold 1x DPBS with Ca2+/Mg2+/0.2% glucose. Transfer the spinal cord to a Petri dish placed on ice packs containing sufficient cold 1x DPBS with Ca2+/Mg2+/0.2% glucose to submerge the entire tissue.</li>
	<li>Carefully remove&#160;the meninges under a microscope within a laminar flow hood. Transfer the spinal cord to an empty Petri dish on ice packs, and using a No. 10 blade surgical scalpel, cut the entire spinal cord into 2-3 mm sections.</li>
</ol>
2. Enzymatic cell dissociation
<ol>
	<li>Add Enzyme mix 1 and Enzyme mix 2 from the commercially available Adult Brain Dissociation Kit following the manufacturer&#39;s protocol (see Table of Materials). Swirl the enzymes in the dish several times to ensure uniform coating of the spinal cord, then incubate at 37 &#176;C for 30 min. Every 5 minutes, gently swirl the dish to prevent tissue clumping and facilitate even distribution of reagents.</li>
	<li>Remove the dish from the incubator and add 350 &#181;L of 0.46 mg/mL DNAse I in Minimal Essential Medium (MEM) (see Table of Materials). Mix by gently swirling.</li>
	<li>Add 1 mL of cold DMEM/0.5% Fetal Bovine Serum (FBS) and mix by gently swirling. Immediately transfer the entire contents to a 5 mL tube.</li>
</ol>
3. Mechanical cell dissociation
<ol>
	<li>Using a P1000 pipette, gently triturate the tissue three times, ensuring that tissue pieces are drawn up each time to further dissociate the tissue. Then, using a plugged 9&#34; glass Pasteur pipette, gently triturate five more times, ensuring no bubbles are generated during the process.</li>
	<li>Place the 5 mL tube upright and allow the tissue to settle at the bottom for 30 s. Once the tissue has settled, draw up the supernatant using a P1000 pipette and pass it through a 30 &#181;m filter into a 15 mL conical tube.</li>
	<li>To the same 5 mL tube with the remaining tissue, add 1 mL of cold DMEM/0.5% FBS. Using a Pasteur pipette, gently triturate three times.</li>
	<li>Allow the tissue to settle at the bottom for 30 s, then pass the supernatant through a 30 &#181;m filter and collect it into the same 15 mL tube as before. Repeat step 3.3 and step 3.4 once more.</li>
	<li>Centrifuge the filtered cell suspension at 300 x g for 10 min at room temperature (RT). Carefully remove and discard the resulting supernatant using a vacuum aspirator.</li>
</ol>
4. Myelin removal
<ol>
	<li>Remove myelin using the debris removal solution (DRS) provided in the Adult Brain Dissociation Kit following the manufacturer&#39;s protocol. After debris removal, add 5 mL of cold DMEM/0.5% FBS to the cell pellet and gently invert the tube three times. Centrifuge the cell suspension at 300 x g for 10 min at room temperature (RT). 
	NOTE: If the cells will be used for in vitro studies and will remain in culture, pre-warmed DMEM/0.5% FBS should be used instead.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 1 mL of DPBS/0.5% FBS (cold for RNA studies and warm for in vitro assays). Count the cells and assess viability using Trypan Blue. 
	NOTE: Cell suspensions from multiple animals may be combined to increase cell concentration.</li>
	<li>Wash the cells by adding DPBS/0.5% FBS up to a total volume of 5 mL. Centrifuge the suspension at 300 x g for 10 min at room temperature and discard the supernatant using a pipette.</li>
</ol>
5. Microglia and astrocyte isolation
<ol>
	<li>Label the cells with anti-CD11b or anti-ACSA-2 microbeads following the manufacturer&#39;s protocol (see Table of Materials).</li>
	<li>Sort the cells by positive selection using the MS columns according to the manufacturer&#39;s instructions (see Table of Materials). After sorting, centrifuge the sorted cells at 300 x g for 10 min and discard the supernatant.</li>
</ol>
6. Plating cells for in vitro assays
NOTE: If cells will not be plated and will be used immediately for RNA analysis, proceed to step 8.
<ol>
	<li>Resuspend the cells in 1 mL of warm DMEM/0.5% FBS. Count the cells and assess their viability using a hemacytometer (see Table of Materials).</li>
	<li>Adjust the cell concentration to 75,000 cells per 50 &#181;L. Add 50 &#181;L of the cell suspension to each Poly-L-lysine-coated coverslip11 in a 24-well plate.</li>
	<li>Incubate at 37 &#176;C for 2 h to allow the cells to adhere to the coverslip.</li>
	<li>Carefully add 450 &#181;L of warm DMEM/5% FBS/Penicillin-Streptomycin (Pen-Strep, see Table of Materials) to each well.</li>
	<li>After 3 days, replace half of the media with 250 &#181;L of warm DMEM/5% FBS/Pen-Strep. For maintenance, completely replace the media with 500 &#181;L of warm DMEM/5% FBS/Pen-Strep every 4-5 days.</li>
</ol>
7. Immunohistochemistry
NOTE: It is best to perform immunohistochemistry analyses after at least 3 days when the media has been replaced at least once. This ensures debris has been removed and cells have completely adhered to the coverslip.
<ol>
	<li>Remove the media and label the cells with anti-mouse mAb O4 (1:100) (see Table of Materials) in warm DMEM/5% FBS/10% normal goat serum (NGS) for 15 min at room temperature (RT) or at 37 &#176;C. 
	NOTE: Skip this step and proceed to step 7.3 if the cells will not be labeled with O4.</li>
	<li>Gently rinse the coverslips by dipping them into warm DMEM/5% FBS three times. Add goat anti-mouse 594 IgM (1: 300) (see Table of Materials) in warm DMEM/5% FBS/10% NGS and incubate at RT for 15 min in the dark. Gently rinse three times in warm DMEM/5% FBS.</li>
	<li>Fix the cells with cold 5% acetic acid/methanol for 15 min at 4 &#176;C. Wash three times with 1x PBS, then label with the appropriate antibody: anti-rabbit GFAP (1: 500), anti-mouse GFAP (1: 500), or anti-rabbit Iba1 (1: 500) in 1% Triton-X100/10% NGS in 1x DPBS (see Table of Materials). Incubate for 1 h at RT. 
	NOTE: Keep coverslips in the dark if they have already been stained with O4.</li>
	<li>Gently rinse three times with PBS. Label with the appropriate secondary antibody: anti-mouse 594 IgG (1: 500), anti-rabbit 488 IgG (1: 500) (see Table of Materials). Incubate for 30 min at RT in the dark.</li>
	<li>Gently rinse three times with PBS. Counterstain with DAPI (1: 1000) for 1 min at RT. Rinse three times with PBS, add mounting medium (see Table of Materials), and mount coverslips onto slides.</li>
</ol>
8. RNA extraction
NOTE: Ideally, there should be at least 100,000 cells to extract sufficient RNA for analysis. If necessary, cells from 2-3 spinal cords may be combined.
<ol>
	<li>Extract RNA using a commercially available RNA isolation kit following the manufacturer&#39;s protocol (see Table of Materials). For each wash step with the RW1 wash buffer provided in the kit, leave the cells in the wash buffer for an additional 2 min.</li>
	<li>Remove any remaining genomic DNA using DNase I according to the manufacturer&#39;s instructions (see Table of Materials). Incubate with DNase at RT for 15 min, then wash with RW1 buffer.</li>
	<li>To increase RNA yield, prior to elution, incubate the samples in RNAse-free water at RT for 10 min. Assess RNA integrity and quantity using a bioanalyzer15 (see Table of Materials).</li>
</ol>

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

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P., 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=Astrocytes+are+necessary+for+blood-brain+barrier+maintenance+in+the+adult+mouse+brain.">Astrocytes are necessary for blood-brain barrier maintenance in the adult mouse brain.</a> Glia. 69 (2), 436-472 (2021).</li><li>Badimon, A., 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=Negative+feedback+control+of+neuronal+activity+by+microglia.">Negative feedback control of neuronal activity by microglia.</a> Nature. 586, 417-423 (2020).</li><li>Yanuck, 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=Microglial+phagocytosis+of+neurons:+diminishing+neuronal+loss+in+traumatic,+infectious,+inflammatory,+and+autoimmune+CNS+disorders.">Microglial phagocytosis of neurons: diminishing neuronal loss in traumatic, infectious, inflammatory, and autoimmune CNS disorders.</a> Front Psychiatry. 10, 712 (2019).</li><li>Xu, Y. J., Au, N. P. B., Ma, C. H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+phenotypic+diversity+of+microglia:+implication+for+microglia-based+therapies+for+alzheimer&#8217;s+disease.">Functional and phenotypic diversity of microglia: implication for microglia-based therapies for alzheimer&#8217;s disease.</a> Front Aging Neurosci. 14, 896852 (2022).</li><li>Song, 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=Microglial-oligodendrocyte+interactions+in+myelination+and+neurological+function+recovery+after+traumatic+brain+injury.">Microglial-oligodendrocyte interactions in myelination and neurological function recovery after traumatic brain injury.</a> J Neuroinflammation. 19 (1), 246 (2022).</li><li>Butler, C. A., 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=Microglial+phagocytosis+of+neurons+in+neurodegeneration,+and+its+regulation.">Microglial phagocytosis of neurons in neurodegeneration, and its regulation.</a> J Neurochem. 158 (3), 621-639 (2021).</li><li>Voskuhl, R. 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=Reactive+astrocytes+form+scar-like+perivascular+barriers+to+leukocytes+during+adaptive+immune+inflammation+of+the+CNS.">Reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the CNS.</a> J Neurosci. 29 (37), 11511-11522 (2009).</li><li>Bordon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFG+activity+in+astrocytes+drives+CNS+inflammation.">MAFG activity in astrocytes drives CNS inflammation.</a> Nature Reviews Immunology. 20, 205 (2020).</li><li>Agalave, N. M., Lane, B. T., Mody, P. H., Szabo-Pardi, T. A., Burton, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+downstream+characterization+of+primary+microglia+and+astrocytes+from+adult+rodent+brain+and+spinal+cord.">Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord.</a> J Neurosci Methods. 340, 108742 (2020).</li><li>Kerstetter, A. E., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+spinal+cord+astrocytes.">Isolation and culture of spinal cord astrocytes.</a> Methods in Molecular Biology. 814, 93-104 (2012).</li><li>Hersbach, B. A., Simon, T., Masserdotti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+neuronal+reprogramming+of+mouse+astrocytes.">Isolation and direct neuronal reprogramming of mouse astrocytes.</a> J Vis Exp. (185), 64175 (2022).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> J Neuroinflammation. 9, 147 (2012).</li><li>Richner, M., Jager, S. B., Siupka, P., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+extrusion+of+the+spinal+cord+and+isolation+of+dorsal+root+ganglia+in+rodents.">Hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents.</a> J Vis Exp. (119), e55226 (2017).</li><li>Davies, J., Denyer, T., Hadfield, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioanalyzer+chips+can+be+used+interchangeably+for+many+analyses+of+DNA+or+RNA.">Bioanalyzer chips can be used interchangeably for many analyses of DNA or RNA.</a> Biotechniques. 60 (4), 197-199 (2016).</li><li>Ahn, J. J., Islam, Y., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+specific+isolation+of+primary+astrocytes+and+microglia+from+adult+mouse+spinal+cord.">Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord.</a> J Neurosci Methods. 375, 109599 (2022).</li><li>Pan, J., Wan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+comparison+of+FACS+and+MACS+isolation+of+enriched+microglia+and+astrocytes+from+mouse+brain.">Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain.</a> J Immunol Methods. 486, 112834 (2020).</li><li>Neuschulz, A., 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+single-cell+RNA+labeling+strategy+for+measuring+stress+response+upon+tissue+dissociation.">A single-cell RNA labeling strategy for measuring stress response upon tissue dissociation.</a> Mol Syst Biol. 19, 11147 (2023).</li><li>CB, 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=One+brain-all+cells:+a+comprehensive+protocol+to+isolate+all+principal+cns-resident+cell+types+from+brain+and+spinal+cord+of+adult+healthy+and+EAE+mice.">One brain-all cells: a comprehensive protocol to isolate all principal cns-resident cell types from brain and spinal cord of adult healthy and EAE mice.</a> Cells. 10 (3), 1-25 (2021).</li></ol>

None

The isolation of pure, viable primary cells is paramount for investigating the structure and function of specific cell types. In the adult mouse, particularly in the spinal cord, this task poses significant challenges, as existing protocols are often not tailored to the adult spinal cord10,17. This protocol presents an efficient and cost-effective method applicable to various downstream applications, including cell culture, flow cytometry, histology, and transcri...

Astrocytes and microglia are versatile glial cells that play vital roles in the central nervous system (CNS), encompassing responsibilities such as regulating neuronal function, contributing to CNS development, maintaining the blood-brain barrier, and participating in other critical processes1,2,3,4. Besides their role in maintaining homeostasis, these glial cells also play a pivotal part in injury and repair mechanisms. Microglia are well-known for their phagocytic, inflammatory, and migratory capabilities following insults or injuries5,6,7. Astrocyte responses in disease are equally diverse, encompassing contributions to inflammation, the formation of glial scars, and the compromise of the blood-brain barrier8,9. Although our understanding of the detrimental and reparative roles of microglia and astrocytes in the CNS has grown, the inherent heterogeneity in both their structure and function necessitates robust tools for studying them in various contexts.
Gaining further insight into the roles of microglia and astrocytes in health and disease requires a combined approach of in vivo and in vitro investigations. In vivo techniques leverage the intricate crosstalk between glial cells and neurons within the CNS, while in vitro methodologies prove valuable when assessing single-cell functions or responses under specific stimuli. Each method offers unique advantages; in vitro studies are essential for understanding the specific roles of these cell types without direct or indirect input from neighboring cells. Additionally, in vitro assays utilizing immortal cell lines present certain benefits, including the ability to proliferate indefinitely, cost-efficiency, and ease of maintenance. However, it&#39;s important to note that primary cells more closely mimic normal physiological responses compared to cell lines. This physiological relevance is crucial in functional assays and transcriptomic analyses.
One of the challenges in obtaining primary cells, particularly from the adult mouse spinal cord, lies in the quantity and viability of the samples. The adult spinal cord, being smaller than the brain and containing a significant amount of myelin, poses unique difficulties. While there are several published protocols detailing the isolation of pure, viable glial cells from neonatal animals or the adult mouse brain10,11,12,13, these methodologies may not be suitable for studying diseases and injuries specific to the spinal cord. In this protocol, we offer a comprehensive procedure to efficiently isolate pure, viable microglia and astrocytes from the adult mouse spinal cord, facilitating downstream applications in cell culture and transcriptomic analyses. This protocol has been successfully employed to isolate these cells from adult mice aged 10 weeks to 5 months, demonstrating its utility across various contexts, including studies involving conditional knockout mice, drug responses, developmental research, and age-related models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,2,2-Tribromoethanol</td>
			<td>Sigma Aldrich</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well tissue culture plate</td>
			<td>Avantor</td>
			<td>10861-558</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methyl-2-butanol, 98%</td>
			<td>Thermo Fisher</td>
			<td>A18304-0F</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>45% glucose solution</td>
			<td>Corning</td>
			<td>25-037-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL capped tubes</td>
			<td>Eppendorf</td>
			<td>30122305</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Adlrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>103-107-677</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ACSA2 Microbead Kit</td>
			<td>Miltenyi</td>
			<td>130-097-679</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Iba1</td>
			<td>Wako</td>
			<td>019-1974</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild-type (WT) mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-093-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Celltrics 30 &#181;m filter</td>
			<td>Sysmex Partec</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chamber (Hemacytometer)</td>
			<td>Hausser Scientific Co</td>
			<td>3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>D4527-40KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>TMO</td>
			<td>15230001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase for RNA purification</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Thermo Fisher</td>
			<td>14040117</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>A5209401</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody (mouse)</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>GFAP antibody (rabbit)</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse 594 IgG</td>
			<td>Invitrogen</td>
			<td>a11032</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse 594 IgM</td>
			<td>Invitrogen</td>
			<td>a21044</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit 488 IgG</td>
			<td>Invitrogen</td>
			<td>a11008</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Iba1 antibody (rabbit)</td>
			<td>Wako</td>
			<td>019-1974</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>MACS Separator</td>
			<td>Miltenyi</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex C/L Pump System</td>
			<td>Thermo Fisher</td>
			<td>77122-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>15-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Adlrich</td>
			<td>439193</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 Antibody</td>
			<td>R&#38;D</td>
			<td>MAB1326</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugged 9&#34; glass pasteur pipette</td>
			<td>VWR</td>
			<td>14672-412</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>74034</td>
			<td></td>
		</tr>
		<tr>
			<td>Royal-tek Surgical scalpel blade no. 10</td>
			<td>Fisher scientific</td>
			<td>22-079-683</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Vein Infusion Set, 23 G x 19 mm</td>
			<td>Kawasumi</td>
			<td>D3K2-23G</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

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

Our research aims to characterize specific cell populations in the central nervous system, specifically the spinal cord, using preclinical models of disease. And we are trying to determine how these cells are affected under pathological conditions and at different time points. It can be challenging to analyze cells, especially in the central nervous system because the cells in the brain and spinal cord form a highly complex matrix and everything is very tightly connected, so it can be difficult to isolate these cells without damaging the system.Our protocol addresses limitations in existing methods for studying spinal cord diseases by efficiently isolating viable microglia astrocytes from the small myelin-rich adult mouse spinal cord. This enables in vitro research on spinal cord related diseases in downstream analysis, filling a crucial research gap. This protocol presents several advantages such as analyzing astrocytes and microglia under pathological conditions and at specific time points.It also uniquely focuses on isolating glial cells from this adult spinal cord alone, excluding brain involvement. This allows researchers to study spinal cord pathologies at the cellular level.

The methods outlined in this protocol enable the isolation of pure and viable microglia and astrocytes from the adult mouse spinal cord, facilitating various downstream applications, including in vitro functional or histological assays and RNA analysis.
A successful isolation for in vitro studies will result in continuous cell proliferation over several days. Adult cells exhibit a slower proliferation rate compared to cells isolated from neonatal animals, and some debris may ...

Watch this Scientific Journal Video about Isolation of Glial Cells from the Adult Mouse Spinal Cord at JoVE.com

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 ...

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Research

JoVE Journal

Neuroscience

Astrocyte and Microglial Cell Isolation from the Adult Mouse Spinal Cord

To begin, remove the spinal cord of a decapitated mouse that has been perfused with PBS. Submerge the spinal cord in cold DPBS in a Petri dish on ice. With an 18-gauge needle and a 10-milliliter syringe filled with cold DPBS, use hydraulic extrusion to isolate the entire spinal cord in a laminar flow hood.Transfer the spinal cord into Petri dish with cold DPBS placed on ice packs. Using a microscope inside the laminar flow hood, carefully remove any visible meninges with sharp forceps. Then, transfer the spinal cord to an empty Petri dish and use a No.10 surgical scalpel blade to cut it into two to three-millimeter-long sections.For enzymatic dissociation of the spinal cells, add Enzyme Mixes 1 and 2 to the spinal cord pieces. Swirl the enzymes in the dish several times to ensure uniform coating of the spinal cord. Incubate the dish at 37 degrees Celsius for 30 minutes, manually swirling the dish every 10 minutes.After incubation, add 350 microliters of DNAse I.Swirl the dish gently to mix. Next, add one milliliter of cold DMEM supplemented with 0.5%FBS before gently mixing. Immediately transfer the entire contents to a five-milliliter tube.With a P1000 pipette, gently triturate the tissue three times. Then, use a plugged nine-inch glass Pasteur pipette to gently triturate the tissue five more times. Place the tube upright for 30 seconds to allow the tissue to settle.With a P1000 pipette, aspirate out the supernatant and transfer it through a 30-micrometer filter into a 15-milliliter conical tube. To the tube with the remaining tissues, add one milliliter of FBS-supplemented DMEM. Then, gently triturate three times with a Pasteur pipette.After letting the tissue settle at the bottom, pass the supernatant through a 30-micrometer filter and collect it into the same 15-milliliter tube used earlier. Centrifuge the filtered cell suspension at 300 g for 10 minutes at room temperature. Use a vacuum aspirator to carefully discard the supernatant.Use the debris removal solution provided in the adult brain dissociation kit to remove the myelin from the cell pellet. Then, add five milliliters of FBS-supplemented DMEM to the pellet and gently invert the tube three times to mix. After centrifuging, discard the supernatant and resuspend the cell pellet in one milliliter of FBS-supplemented DPBS.Centrifuge again before discarding the supernatant. Labeled the cells with Anti-ACSA-2 MicroBeads following the manufacturer's protocol. Use the magnetic separation columns to separate the cells by positive selection.Then, centrifuge the cells at 300 g for 10 minutes before discarding the supernatant. Next, resuspend the cell pellet in one milliliter of warm FBS-supplemented DMEM. Transfer the cell suspension to a hemocytometer to count the cells and assess their viability.Adjust the cell concentration to 75, 000 cells per 50 microliters with FBS DMEM. Then, transfer 50 microliters of the cell suspension to a poly-l-lysine-coated coverslip in a 24-well plate. Incubate the plate at 37 degrees Celsius for two hours to allow the cells to adhere to the coverslip.Then, carefully add 450 microliters of warm and antibiotic-supplemented FBS DMEM to each well. Successful isolation and proliferation of microglia and astrocytes were observed. By day four, astrocytes were observed to form longer processes, while oval microglial cells with shorter spindles were seen.The astrocytes formed a connected confluent layer by day seven, with the microglia displaying fewer and shorter processes. Cell sorting confirmed 92 to 93%cell viability of the isolated cell culture.

1.2K Views.  The George Washington University School of Medicine.  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.

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

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