We thank Kathy Gage for her excellent editorial contribution. The illustration&#160;figures were&#160;created with BioRender.com. This study was supported by funds from the Department of Anesthesiology (Duke University Medical Center) and NIH grants NS099590, HL157354, and NS127163.

The protocol was approved by the Duke Institute Animal Care and Use Committee (IACUC). Male C57Bl/6 mice (3-4 months old; 22-28 g) were used in the current study. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Single-cell suspension from mouse brain
NOTE: Figure 1 illustrates the overview of the brain cell isolation protocol.
<ol>
	<li>Anesthetize, intubate...

Obtaining Single-Cell Suspensions from Murine Brain and Skull Bone Marrow

<ol>
	<li>Bohr, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glymphatic+system:+Current+understanding+and+modeling.">The glymphatic system: Current understanding and modeling.</a> iScience. 25 (9), 104987 (2022).</li><li>Jiang-Xie, L. F., 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=Neuronal+dynamics+direct+cerebrospinal+fluid+perfusion+and+brain+clearance.">Neuronal dynamics direct cerebrospinal fluid perfusion and brain clearance.</a> Nature. 627 (8002), 157-164 (2024).</li><li>Goertz, J. E., Garcia-Bonilla, L., Iadecola, C., Anrather, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+compartments+at+the+brain's+borders+in+health+and+neurovascular+diseases.">Immune compartments at the brain's borders in health and neurovascular diseases.</a> Semin Immunopathol. 45 (3), 437-449 (2023).</li><li>Herisson, F., 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=Direct+vascular+channels+connect+skull+bone+marrow+and+the+brain+surface+enabling+myeloid+cell+migration.">Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration.</a> Nat Neurosci. 21 (9), 1209-1217 (2018).</li><li>Mazzitelli, J. 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=Skull+bone+marrow+channels+as+immune+gateways+to+the+central+nervous+system.">Skull bone marrow channels as immune gateways to the central nervous system.</a> Nat Neurosci. 26 (12), 2052-2062 (2023).</li><li>Cugurra, 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=Skull+and+vertebral+bone+marrow+are+myeloid+cell+reservoirs+for+the+meninges+and+CNS+parenchyma.">Skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and CNS parenchyma.</a> Science. 373 (6553), eabf7844 (2021).</li><li>Brioschi, 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=Heterogeneity+of+meningeal+b+cells+reveals+a+lymphopoietic+niche+at+the+CNS+borders.">Heterogeneity of meningeal b cells reveals a lymphopoietic niche at the CNS borders.</a> Science. 373 (6553), eabf9277 (2021).</li><li>Smyth, L. C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+direct+connections+between+the+dura+and+the+brain.">Identification of direct connections between the dura and the brain.</a> Nature. 627, 165-173 (2024).</li><li>Mazzitelli, J. 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=Cerebrospinal+fluid+regulates+skull+bone+marrow+niches+via+direct+access+through+dural+channels.">Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels.</a> Nat Neurosci. 25 (5), 555-560 (2022).</li><li>Pulous, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+can+exit+into+the+skull+bone+marrow+and+instruct+cranial+hematopoiesis+in+mice+with+bacterial+meningitis.">Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis.</a> Nat Neurosci. 25 (5), 567-576 (2022).</li><li>Li, 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=Mouse+cardiac+arrest+model+for+brain+imaging+and+brain+physiology+monitoring+during+ischemia+and+resuscitation.">Mouse cardiac arrest model for brain imaging and brain physiology monitoring during ischemia and resuscitation.</a> J Vis Exp. (194), e65340 (2023).</li><li>Wang, W., 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=Development+and+evaluation+of+a+novel+mouse+model+of+asphyxial+cardiac+arrest+revealed+severely+impaired+lymphopoiesis+after+resuscitation.">Development and evaluation of a novel mouse model of asphyxial cardiac arrest revealed severely impaired lymphopoiesis after resuscitation.</a> J Am Heart Assoc. 10 (11), e019142 (2021).</li><li>Li, X., 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=Single-cell+transcriptomic+analysis+of+the+immune+cell+landscape+in+the+aged+mouse+brain+after+ischemic+stroke.">Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.</a> J Neuroinflammation. 19 (1), 83 (2022).</li><li>Wang, Y. C., 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=Perk+(protein+kinase+RNA-like+er+kinase)+branch+of+the+unfolded+protein+response+confers+neuroprotection+in+ischemic+stroke+by+suppressing+protein+synthesis.">Perk (protein kinase RNA-like er kinase) branch of the unfolded protein response confers neuroprotection in ischemic stroke by suppressing protein synthesis.</a> Stroke. 51 (5), 1570-1577 (2020).</li><li>Posel, C., Moller, K., Boltze, J., Wagner, D. C., Weise, 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+flow+cytometric+analysis+of+immune+cells+from+the+ischemic+mouse+brain.">Isolation and flow cytometric analysis of immune cells from the ischemic mouse brain.</a> J Vis Exp. 108, e53658 (2016).</li><li>Srakocic, 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=Proposed+practical+protocol+for+flow+cytometry+analysis+of+microglia+from+the+healthy+adult+mouse+brain:+Systematic+review+and+isolation+methods'+evaluation.">Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic review and isolation methods' evaluation.</a> Front Cell Neurosci. 16, 1017976 (2022).</li><li>Disano, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+central+nervous+system+tissues+and+associated+meninges+for+the+downstream+analysis+of+immune+cells.">Isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells.</a> J Vis Exp. 159, e61166 (2020).</li><li>Mattei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+induces+transcriptional+and+proteotype+bias+in+brain+cell+populations.">Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations.</a> Int J Mol Sci. 21 (21), (2020).</li><li>Mcgill, C. J., Lu, R. J., Benayoun, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+analysis+of+mouse+neutrophil+netosis+by+flow+cytometry.">Protocol for analysis of mouse neutrophil netosis by flow cytometry.</a> STAR Protoc. 2 (4), 100948 (2021).</li><li>Su, Y., 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=Meningeal+immunity+and+neurological+diseases:+New+approaches,+new+insights.">Meningeal immunity and neurological diseases: New approaches, new insights.</a> J Neuroinflammation. 20 (1), 125 (2023).</li><li>Niu, C., 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=Mechanical+isolation+of+neonatal+and+adult+mouse+dura+leukocytes+for+flow+cytometry+analysis.">Mechanical isolation of neonatal and adult mouse dura leukocytes for flow cytometry analysis.</a> STAR Protoc. 4 (2), 102272 (2023).</li><li>Roussel-Queval, A., Rebejac, J., Eme-Scolan, E., Paroutaud, L. A., Rua, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+immunohistochemistry+of+the+mouse+dural+meninges+for+immunological+and+virological+assessments.">Flow cytometry and immunohistochemistry of the mouse dural meninges for immunological and virological assessments.</a> STAR Protoc. 4 (1), 102119 (2023).</li><li>Louveau, A., Filiano, A. J., Kipnis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meningeal+whole+mount+preparation+and+characterization+of+neural+cells+by+flow+cytometry.">Meningeal whole mount preparation and characterization of neural cells by flow cytometry.</a> Curr Protoc Immunol. 121 (1), e50 (2018).</li></ol>

Here, two simple yet effective protocols are presented for isolating immune cells from the brain and skull bone marrow. These protocols can reliably yield a large quantity of viable immune cells that may be suitable for diverse downstream applications, in particular for flow cytometry.
To study neuroinflammation in various brain disorders, many protocols for immune cell preparations from the brain have been established and used in different laboratories15,<su...

The brain, the central hub of the nervous system, is protected by the skull. Beneath the skull are three layers of connective tissue known as the meninges - the dura mater, arachnoid mater, and pia mater. Cerebrospinal fluid (CSF) circulates in the subarachnoid space between the arachnoid mater and pia mater, cushioning the brain and also removing waste via the glymphatic system1,2. Together, this unique architecture provides a secure and supportive environment that maintains the stability of the brain and shields it from potential injury.
The brain has long been considered immune-privileged. However, this notion has been partially abandoned as mounting evidence indicates that, in addition to resident microglia in the parenchyma, the borders of the brain, including the choroid plexus and meninges, host a diverse array of immune cells3. These cells play critical roles in maintaining homeostasis, surveilling brain health, and initiating the immune response to brain injury. Notably, recent findings indicate that the skull is involved in meninges immunity, and may contribute to the immune response in the brain after injury. In 2018, Herisson et al. made a seminal discovery of direct vascular channels that link the skull bone marrow to the meninges, thereby establishing an anatomical route for leukocyte migration4,5. Later, Cugurra et al. demonstrated that many myeloid cells (e.g., monocytes and neutrophils) and B cells in the meninges do not originate from the blood6. Using techniques such as calvaria bone-flap transplantation and selective irradiation regimens, the authors provided compelling evidence that the skull bone marrow serves as a local source for myeloid cells in the meninges as well as CNS parenchyma after CNS injury6. Further, another study proposed that meningeal B cells are constantly supplied by the skull bone marrow7. More recently, a novel structure, termed the arachnoid cuff exit (ACE), has been identified as a direct gateway between the dura mater and the brain for immune cell trafficking8.
These exciting findings have important implications for the origin of infiltrating immune cells into the injured brain (e.g., after ischemic stroke). A large body of evidence has indicated that after stroke, many immune cells infiltrate the brain, contributing to both acute brain damage and chronic brain recovery. The conventional notion&#160;is that these cells are circulating leukocytes in the blood that infiltrate the brain, which is largely facilitated by stroke-induced blood-brain barrier damage. However, this notion has been challenged. In one study, immune cells in the skull and the tibia of mice were labeled differently, and at 6 h after stroke, a significantly greater decrease in neutrophils and monocytes was found in the skull vs. the tibia, and more skull-derived neutrophils were present in the ischemic brain. These data suggest that in the acute stroke phase, neutrophils in the ischemic brain primarily originate from the skull bone marrow4. Interestingly, CSF may guide this migration. Indeed, two recent reports demonstrated that CSF can directly relay signaling cues from the brain into the skull bone marrow via skull channels, and instruct cell migration and hematopoiesis in the skull bone marrow after CNS injury9,10.
In light of these recent findings, it has become important to analyze changes in immune cells in both the brain and the skull bone marrow, when studying the immune response to brain disorders. In such investigations, sufficient numbers of high-quality immune cells are needed for downstream analyses such as cell sorting, flow cytometry analysis, and single-cell RNA sequencing (scRNA-seq). Here, the overall goal is to present two optimized procedures for preparing single-cell suspensions from brain tissue and skull bone marrow. It is important to note that the calvaria (frontal bone, occipital bone, and parietal bones) of the skull are typically used to extract bone marrow, and this bone marrow is specifically referred to as skull bone marrow throughout this study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL microcentrifuge tubes</td>
			<td>VWR</td>
			<td>76332-066</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>VWR</td>
			<td>76332-068</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G x 1 in BD PrecisionGlide Needle</td>
			<td>BD Biosciences</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well V-bottom microplate&#160;</td>
			<td>SARSTEDT</td>
			<td>82.1583</td>
			<td></td>
		</tr>
		<tr>
			<td>AURORA&#160; flow cytometer</td>
			<td>Cytek bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher</td>
			<td>BP9706-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-AF594</td>
			<td>BioLegend</td>
			<td>101254</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD19-BV785</td>
			<td>BioLegend</td>
			<td>115543</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD19-FITC</td>
			<td>BioLegend</td>
			<td>115506</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD3-APC</td>
			<td>BioLegend</td>
			<td>100312</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>BioLegend</td>
			<td>100206</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD45-Alex 700</td>
			<td>BioLegend</td>
			<td>103128</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>CD45-BV421</td>
			<td>Biolegend</td>
			<td>103133</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Cell Strainer 70 um</td>
			<td>Avantor</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps&#160;</td>
			<td>V. Mueller</td>
			<td>NL1410</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc Block</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5047</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>N7167</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Ly6G-BV421</td>
			<td>BioLegend</td>
			<td>127628</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Ly6G-PerCp-cy5.5</td>
			<td>BioLegend</td>
			<td>127615</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>NK1.1-APC-cy7</td>
			<td>BioLegend</td>
			<td>108723</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Percoll (density gradient medium)</td>
			<td>Cytiva</td>
			<td>17089101</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (10x)</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer (10x)</td>
			<td>BioLegend</td>
			<td>420302</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>SKLAR</td>
			<td>64-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>WHEATON Dounce Tissue, 15 mL Size</td>
			<td>DWK Life Sciences</td>
			<td>357544</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating immune cells from mouse brain and skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

Isolating Immune Cells from Mouse Brain and Skull

Our research focuses on the complex immune response following brain ischemia caused by ischemic stroke and cardiac arrest. Brain ischemia triggers the activation resident immune cells in the brain and the infiltration of immune cells. However, our understanding of this immune cells remains limited.We aim to dissect the immune state, origin, and functional roles of this brain cells after ischemia. Recent research highlights the surprising role of the skull in the brain's immune response following injury. Therefore, it is important to analyze changes in immune cells in both the brain tissue and the skull bone marrow after brain ischemia.To this end, a critical step is to obtain a sufficient number of high-quality immune cells for further analysis. Our protocols enable researchers to rapidly obtain large quantities of available immune cells from the brain and the skull bone marrow. The brain immune cell protocol uses a mechanical dissociation approach at low temperature throughout, ensuring better preservation of the transcriptional and the proteomic profiles.The skull bone marrow protocol is simple, yet effective for extracting bone marrow cells from the skull. We'll continue our comprehensive research on the impact of brain ischemia on immune cells in brain tissue and the skull. Given that neuroinflammation plays a key role in the pathophysiology of brain injury after ischemia, our research is expected to discover novel therapeutic targets for brain protection after ischemic stroke or cardiac arrest.

To prepare immune cells from the mouse brain tissue, the protocol generally yields cells with high viability (84.1%&#160;&#177; 2.3% [mean&#160;&#177; SD]). Approximately 70%-80% of these cells are CD45 positive. In the normal mouse brain, nearly all CD45+ cells are microglia (CD45LowCD11b+), as expected. This protocol has been used in the laboratory for various applications, including flow cytometry analysis, fluorescence-activated cell sorting (FACS), and scRNA-seq analysis. As an examp...

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...