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

<ol><li>Abbott, N. J., Rönnbäck, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte-endothelial+interactions+at+the+blood-brain+barrier%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Astrocytes+are+necessary+for+blood-brain+barrier+maintenance+in+the+adult+mouse+brain%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Negative+feedback+control+of+neuronal+activity+by+microglia%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Microglial+phagocytosis+of+neurons%3A+diminishing+neuronal+loss+in+traumatic%2C+infectious%2C+inflammatory%2C+and+autoimmune+CNS+disorders%5BTitle%5D)+AND+%22Front+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((Functional+and+phenotypic+diversity+of+microglia%3A+implication+for+microglia-based+therapies+for+alzheimer%E2%80%99s+disease%5BTitle%5D)+AND+%22Front+Aging+Neurosci%22%5BJournal%5D)">Functional and phenotypic diversity of microglia: implication for microglia-based therapies for alzheimer’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/pubmed?term=((Microglial-oligodendrocyte+interactions+in+myelination+and+neurological+function+recovery+after+traumatic+brain+injury%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">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/pubmed?term=((Microglial+phagocytosis+of+neurons+in+neurodegeneration%2C+and+its+regulation%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">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/pubmed?term=((Reactive+astrocytes+form+scar-like+perivascular+barriers+to+leukocytes+during+adaptive+immune+inflammation+of+the+CNS%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((MAFG+activity+in+astrocytes+drives+CNS+inflammation%5BTitle%5D)+AND+%22Nature+Reviews+Immunology%22%5BJournal%5D)">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/pubmed?term=((Isolation%2C+culture%2C+and+downstream+characterization+of+primary+microglia+and+astrocytes+from+adult+rodent+brain+and+spinal+cord%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+culture+of+spinal+cord+astrocytes%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+direct+neuronal+reprogramming+of+mouse+astrocytes%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">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/pubmed?term=((Hydraulic+extrusion+of+the+spinal+cord+and+isolation+of+dorsal+root+ganglia+in+rodents%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Bioanalyzer+chips+can+be+used+interchangeably+for+many+analyses+of+DNA+or+RNA%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((Cell+type+specific+isolation+of+primary+astrocytes+and+microglia+from+adult+mouse+spinal+cord%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Methodological+comparison+of+FACS+and+MACS+isolation+of+enriched+microglia+and+astrocytes+from+mouse+brain%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">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/pubmed?term=((A+single-cell+RNA+labeling+strategy+for+measuring+stress+response+upon+tissue+dissociation%5BTitle%5D)+AND+%22Mol+Syst+Biol%22%5BJournal%5D)">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/pubmed?term=((One+brain-all+cells%3A+a+comprehensive+protocol+to+isolate+all+principal+cns-resident+cell+types+from+brain+and+spinal+cord+of+adult+healthy+and+EAE+mice%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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><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&#039;,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&amp;nbsp;</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 &amp;micro;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&#039;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&amp;amp;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&quot; 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.

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

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.

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 ...

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1.8K 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.

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

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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

Immunology and Infection

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

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

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

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

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

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

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.

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.

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

Isolation of Microglia from the Cortex of an Adult Mouse Brain

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

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

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

A Simple Technique to Isolate and Culture Murine Astrocytes

Source: Wolfes, A. C., et al., <a href="https://app.jove.com/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
...

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

Source: Schildge, S. et al. <a href="https://www.jove.com/v/50079/isolation-and-culture-of-mouse-cortical-astrocytes">Isolation and Culture of Mouse Cortical Astrocytes.</a> J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.

Source: Schildge, S. et al. Isolation and Culture of Mouse Cortical Astrocytes. J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure ...