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, and secure the mouse, as demonstrated previously11.</li>
	<li>Make an upper abdominal incision through the skin and underlying muscle layers, and carefully cut through the diaphragm and ribs to create the opening into the thoracic cavity.</li>
	<li>Fill a 30 mL syringe with cold PBS and attach it to the blunt perfusion needle via silicone tubing. Carefully insert the perfusion needle into the left ventricle.</li>
	<li>Perform a small nick at the right atrium to allow blood drainage and then perfuse12 the mouse at a flow rate of ~ 10 mL/min for 3-4 min. 
	NOTE: To improve blood clearance in the brain, increasing the volume of perfusion solution and adding heparin may be considered.</li>
	<li>Decapitate the mouse and make a midline incision through the skin on the top of the head to expose the skull.</li>
	<li>Carefully scrape away any connective tissue overlying the skull using blunt forceps.</li>
	<li>Use sharp dissection scissors to carefully cut through the skull sutures along the midline and on both sides, creating a flap.</li>
	<li>Gently lift the skull flap to expose the brain, and use blunt forceps to carefully remove the brain from the cranial cavity.</li>
	<li>Immerse the brain in a Petri dish containing ice-cold PBS and remove any remaining meninge tissues. 
	NOTE: To better preserve cell viability, it is recommended to perform all following steps on ice or at 4 &#176;C.</li>
	<li>Place the brain in a pre-cooled 15 mL glass Dounce homogenizer containing 20 mL (whole brain) or 12 mL (half brain) of ice-old HBSS buffer.</li>
	<li>Use the loose Dounce pestle to gently and slowly dissociate the brain tissue with around 100-120 strokes on ice. 
	NOTE: This step is critical to obtaining a large quantity of healthy brain cells. This step takes approximately 10 min. Stop homogenization when no substantial brain tissue chunks are visible in the suspension. At this point, the pestle can easily move down without any force.</li>
	<li>Filter the resulting brain tissue suspension through a 70 &#181;m cell strainer in a 50 mL centrifuge tube.</li>
	<li>Rinse the Dounce tube with 5 mL HBSS, and proceed with filtration to maximize the cell collection.</li>
	<li>Centrifuge the filtered tissue suspension at 550 x g for 6 min at 4 &#176;C.</li>
	<li>Remove the supernatant using a vacuum aspirator, and resuspend the cell pellet in 1 mL of 30% isotonic density gradient solution. 
	NOTE: To make 15 mL of 30% isotonic density gradient solution, mix 4.5 mL of 100% stock density gradient medium, 1.5 mL of 10&#215; PBS, and 9 mL of ddH2O.</li>
	<li>Transfer the cell suspension into a 15 mL centrifuge tube, and add 9 mL (whole brain) or 6 mL (half brain) of 30% density gradient solution.</li>
	<li>Gently invert the tube to ensure thorough mixing.</li>
	<li>Immediately centrifuge the tube at 845 x g with acceleration and brake set at level 3, for 20 min at 4 &#176;C.</li>
	<li>Gently remove the tube from the centrifuge without shaking, and carefully aspirate the upper myelin layer using a 1 mL tip connected to the vacuum. 
	NOTE: It is critically important not to disturb this layer. Any remaining myelin can negatively impact cell health and compromise downstream analyses.</li>
	<li>Aspirate the supernatant, leaving 1-2 mL behind, and resuspend the cell pellet with a minimum of 10 mL cold HBSS.</li>
	<li>Centrifuge at 550 x g for 6 min at 4 &#176;C.</li>
	<li>Wash one more time with a minimum of 7 mL of cold HBSS.</li>
	<li>Remove as much of the supernatant as possible, and resuspend the cells for flow cytometry analysis using 100-200 &#181;L of flow cytometry buffer. 
	NOTE: The flow cytometry FACS buffer used for this study contains 0.5% BSA and 2 mM EDTA in 1&#215; PBS. A 96-well V-bottom plate for cell staining is recommended to minimize cell loss during the washing steps.</li>
	<li>Perform flow cytometry analysis using a standard protocol established in individual laboratories13.
	<ol>
		<li>Briefly, mix 80 &#181;L of cell suspension with 20 &#181;L of the antibody mixture in a 96-well V-bottom plate and incubate for 25 min at 4 &#176;C.</li>
		<li>Wash with 200 &#181;L of FACS buffer and then resuspend the cells with 100 &#181;L of HBSS plus cell viability staining reagent.</li>
		<li>After a 15 min incubation at 4 &#176;C, wash with FACS buffer and resuspend the cells in 200 &#181;L of FACS buffer for flow cytometry analysis.</li>
	</ol>
	</li>
</ol>
2. Preparation of bone marrow single-cell suspension from mouse calvaria
NOTE: Figure 2 depicts the overview of the skull bone marrow isolation procedure.
<ol>
	<li>Perfuse the mouse as described in step 1.4.</li>
	<li>Decapitate the mouse and make a midline skin incision on the scalp to expose the skull.</li>
	<li>Cut through the skull, starting from the foramen magnum and moving forward along the lateral sides of the calvaria using sharp scissors. Then, lift and remove the calvaria. 
	NOTE: The brain can be harvested from the same mouse and processed using step 1, enabling analysis of immune cells from both the brain and skull bone marrow.</li>
	<li>Carefully peel off the dura mater from the skull under a dissecting microscope using blunt forceps. 
	NOTE: It is easier to start at the edges of the occipital bone, slowly detach, and progress steadily towards the frontal area, working inward.</li>
	<li>Place the calvaria in the dish on ice and use a 1 mL pipette to rinse the calvaria, ensuring the removal of blood residues from its surface.</li>
	<li>Transfer the calvaria to a new dish with fresh, cold PBS sufficient to immerse the calvaria.</li>
	<li>Use sterile scissors to cut the calvaria into approximately 3 mm x 3 mm fragments in cold PBS. 
	NOTE: Cropping methods may affect the efficiency of calvaria bone marrow extraction. It is recommended that the same cropping method be used to minimize variability. It is also important to cut open the sutures. Figure 2B illustrates one approach.</li>
	<li>Poke a hole in the bottom of a 500 &#181;L microcentrifuge tube using an 18 G needle.</li>
	<li>Insert this tube into a 1.5 mL microcentrifuge tube containing 20 &#181;L of cold PBS.</li>
	<li>Transfer the calvaria fragments into the 500 &#181;L tube. 
	NOTE: The bone fragments should be loosely packed to increase extraction efficiency.</li>
	<li>Centrifuge at 10,000 x g for 30 s at 4 &#176;C. 
	NOTE: To maximize cell recovery, use an 18 G needle to gently stir the calvaria fragments, and repeat this step 1-2 more times.</li>
	<li>Resuspend the collected calvaria bone marrow cells in 500 &#181;L of Red Blood Cell lysis buffer and incubate at room temperature for 30 s.</li>
	<li>Transfer the suspension to a 15 mL centrifuge tube containing 4 mL of cold PBS.</li>
	<li>Centrifuge at 400 x g for 6 min at 4 &#176;C.</li>
	<li>Wash one more time with 5 mL of cold PBS.</li>
	<li>Resuspend the cell pellet in an appropriate buffer. For flow cytometry, we typically use 500 &#181;L of FACS buffer.</li>
	<li>For flow cytometry analysis, perform cell counting and use approximately 1 x 106 cells for antibody staining. 
	NOTE: Ensure to include the Fc block step before antibody staining13.</li>
</ol>

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), 7844 (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), 9277 (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.

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...

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

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.

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 ...

isolating-immune-cells-from-mouse-brain-and-skull

Research

JoVE Journal

Neuroscience

2.3K Views.  Duke University Medical Center. 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.

Video: Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...