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 <strong cla...

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

<ol>
	<li>Abbott, N. J., R&#246;nnb&#228;ck, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nat Rev Neurosci. 7, 41-53 (2006).</li><li>Heithoff, B. 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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Neuroscience

1.1K ビュー.  The George Washington University School of Medicine. 私たちの研究は、疾患の前臨床モデルを用いて、中枢神経系、特に脊髄における特定の細胞集団を特徴づけることを目的としています。そして、これらの細胞が病理学的条件下で、さまざまな時点でどのように影響を受けるかを決定しようとしています。特に中枢神経系では、脳と脊髄の細胞は非常に複雑なマトリックスを形成し、すべてが非常に緊密に接続されてい?...