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 laboratories15,16,17. The common approaches involve enzyme-based cell dissociation followed by gradient separation. Typically, in these protocols, brain tissue is minced and then incubated in an enzyme digestion solution containing both collagenase and DNase for 30-60 min at 37 &#176;C with gentle shaking. After digestion, cells undergo further dissociation using a mechanical method such as pipetting or Dounce homogenization. After passing through a cell strainer (e.g., 70 &#181;m strainer), cells are then subjected to gradient centrifugation (often 30%/70% density gradient solution) to remove myelin, red blood cells, and debris and to enrich leukocytes. However, this approach requires a prolonged enzymatic incubation at 37 &#176;C that may substantially alter the transcriptome and proteome of brain immune cells. Indeed, a systematic investigation suggested that the enzymatic approach introduces profound alterations in the transcriptome and proteotype of specific brain cells18. This could lead to artifacts in subsequent transcriptomic analyses such as scRNA-seq. In contrast, the protocol reported here uses a mechanical dissociation approach at cold temperatures throughout the procedure, thus suppressing cellular metabolism. This ensures better preservation of the transcriptional and proteomic state, minimizing potential artifacts. During protocol development, we noticed that using a large Dounce homogenizer for homogenizing brain tissue is critical to achieving higher cell counts with lower cell death. It is likely that during Dounce homogenization, the massive presence of extracellular matrices, cell debris, and cytoplasmic components is toxic to cells. Using an excessive amount of buffer helps to dilute these toxic substances and, thus, mitigates their harmful effects, leading to better cell viability. The flow cytometry data confirmed an approximate 85% viability rate of immune cells prepared with this protocol. Moreover, it is notable that centrifugation with only a 30% density gradient solution effectively enriches brain immune cells and removes myelin. This can also reduce the cost of cell preparation.
Studies on immune cells in the skull bone marrow are still limited. A method commonly used to isolate cells from the mouse skull calvarium involves cutting the skull into small pieces and then mechanically crushing the pieces with a pestle6. In the current protocol, brief centrifugation is used. This method is adapted from a published protocol for preparing bone marrow from femurs and tibias19. For these long bones, one needs only to cut off one end of the bone to collect bone marrow by centrifugation. For the skull, this step is modified by cutting it into several small pieces to open the marrow cavity. It is recommended to use a consistent pattern for cutting the skull in order to obtain reproducible cell counts. Moreover, to maximize the recovery of bone marrow cells, the skull pieces can undergo 2-3 rounds of centrifugation with mixing between each centrifugation.
Both protocols have limitations. First, the use of 30% density gradient solution centrifugation may not effectively remove the remaining red blood cells in the brain, which may occur due to incomplete perfusion. Moreover, if myelin structure is modulated under certain conditions, a 30% density gradient solution may not be an optimal concentration for their removal. In this case, another working concentration of density gradient solution needs to be established. Second, although flow cytometry analysis indicates good cell viability of the brain immune cells using the described protocol, it still needs to be evaluated for other applications, such as transcriptomic analysis in which actinomycin D may be used. Third, the calvaria is used to prepare skull bone marrow. However, it must be noted that immune cells exist in many other parts of the skull. Lastly, the meninges, especially the dura mater, harbor diverse immune cells and play roles in brain homeostasis and dysfunction20. For immune analysis of the meninges, a protocol published elsewhere may be adopted17,21,22,23.
In summary, two robust protocols for isolating immune cells from both the mouse brain and skull are described. These protocols are expected to be useful for obtaining a large quantity of high-quality cells, suitable for various downstream applications.

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><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&amp;nbsp;</td><td>SARSTEDT</td><td>82.1583</td><td></td></tr><tr><td>AURORA&amp;nbsp; 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&amp;nbsp;</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 example, a flow cytometry analysis was conducted in a stroke model (Figure 3). Mice were subjected to a transient stroke model, 30 min filament middle cerebral artery occlusion (MCAO)14. On day 3 after stroke, cells were prepared from mouse brains and stained with common immune cell surface markers. The gating strategy is depicted in Figure 3A. Consistent with prior research13,15, a marked increase in CD45High cells in the ipsilateral hemisphere was observed (Figure 3B,C), indicating infiltration of the immune cell into the brain.
For the skull bone marrow protocol, a substantial yield of skull bone marrow cells (around 2 x 106 cells) was consistently achieved with excellent viability (93.8% &#177;&#160;1.8% [mean&#160;&#177; SD]). As expected, most of these cells are CD45 positive. To illustrate the utility of this protocol, a comparative study was performed to characterize the immune cell composition in both the femur and skull bone marrow of naive mice (Figure 4). As depicted in the representative plots, the immune cell composition was similar between the femur and skull bone marrow (Figure 4A,B). Interestingly, the frequency of neutrophils appears to be higher in the femur than in the skull bone (Figure 4C). However, this finding requires further validation, as the sample size was small for this pilot study.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66861/66861fig01.jpg" /> 
Figure 1: Overview of the brain cell isolation protocol. <a href="https://www.jove.com/files/ftp_upload/66861/66861fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66861/66861fig02.jpg" /> 
Figure 2: The skull bone marrow cell isolation protocol. (A) Overview of the procedure. (B) The recommended pattern for skull cutting. <a href="https://www.jove.com/files/ftp_upload/66861/66861fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66861/66861fig03.jpg" /> 
Figure 3: Representative flow cytometry analysis of the immune cells in the stroke brain. (A) An example of the gating strategy. The plots depict the following immune cell populations: microglia (CD45LowCD11b+), NK cells (CD45HighNK1.1+), neutrophils (CD45HighLy6G+), T cells (CD45HighCD11b-CD3+), and B cells (CD45HighCD11b-CD19+). This example represents an analysis of brain immune cells in the ipsilateral hemisphere on day 3 after stroke. (B,C) Infiltrating immune cells into the stroke brain. Young male mice were subjected to transient middle cerebral artery occlusion (MCAO). On day 3, brains were collected and split into left (contralateral) and right (ipsilateral) hemispheres. Brain cells were prepared according to the described protocol and then subjected to flow cytometry analysis. Representative plots illustrate increased infiltrating immune cells into the ipsilateral hemisphere (CD45High cells; B). Quantitative data are shown in the bar graphs (C). The cell numbers were calculated based on the cell count and volume recorded in the flow cytometry data. The data are presented as mean &#177; SEM. *p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/66861/66861fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66861/66861fig04.jpg" /> 
Figure 4: Representative flow cytometry analysis of the skull bone marrow cells. For comparison, analysis of bone marrow cells from the femur was included. The plots depict the following immune cell populations: NK cells (CD45+NK1.1+), neutrophils (CD45+CD11b+Ly6G+), T cells (CD45+CD11b-CD3+), and B cells (CD45+CD11b-CD19+). (A) Representative plots for femur bone marrow. (B) Representative plots for skull bone marrow. (C) Comparison of the immune cell composition between femur and skull bone marrow. The data are presented as mean &#177; SEM. *p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/66861/66861fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Immunology and Infection

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 ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

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 of Hypothalamic Microglia from Mice Using MACS for In Vitro Assays

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

Author Spotlight: Investigating the Impact of Nutrition on Mouse Brain Function and Metabolic Disorders 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

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

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app.jove.com/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

Co-culture and Interaction Studies

Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

Source: P&#246;sel, C., et al. <a href="https://app.jove.com/v/53658">Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for isolating brain cells and infiltrated immune cells from ischemic mouse brain tissues by enzymatic digestion, followed by the removal of cell debris and myelin by density gradient centrifugation, to study inflammation responses in stroke research.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preparation of Reagents

	Digestion ...

Neuropathology

Cerebrovascular Diseases

Preparing a Single-Cell Suspension of Immune Cells from Murine Brain Tissue

Source: DiSano, K. D., et al. <a href="https://app.jove.com/v/61166">Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune Cells.</a> J. Vis. Exp. (2020).
The video demonstrates the preparation of a single-cell suspension of immune cells from a murine brain tissue. It involves enzymatic digestion to release cells from the brain tissue, followed by density gradient centrifugation to isolate the immune cells based on their densities to obtain the single-cell suspension.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparing single-cell ...

Isolating Immune Cells from Mouse Choroid Plexuses

Source: Dominguez-Belloso, et al. <a href="https://app.jove.com/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...