Figures were created using Adobe Illustrator (version 2023) and Servier Medical Art (https://smart.servier.com). Antonia Henes was supported by J&#252;rgen Manchot Stiftung.

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

All authors declare to have no conflicts of interest.

So far, methods to map CNS-resident cells ex vivo by combining mass spectrometry and RNA sequencing offer a very precise cellular profiling in health and disease but require ambitious technical knowledge and expertise in this field50,51. Further, they do not allow functional analyses and are very expensive. Besides that, microfluidic brain-on-a-chip systems provide a rapid and affordable screening for disease mechanisms and testing of new therapeutic approaches with the restriction of cell growth and migration52,53,54,55. CNS organoids could also represent an equivalent alternative in the future for the investigation of cellular modeling, inter-cellular connections, and interactions during disease courses56,57,58,59. However, fluorescence- and magnetic-activated cell sorting are currently the most effective methods to generate pure and viable single-cell suspensions ex vivo35,60,61. Even if other established manufactural protocols for the isolation of CNS-resident cell types are similar regarding the individual steps of the magnetic isolation and the prior cell dissociation, they are intended to be performed for each cell type separately. By contrast, the current protocol integrates different isolation methods for each CNS-resident cell type into a logical context so that they can be performed simultaneously at once and from one single CNS cell suspension (Table 1, Table 2). Thus, it enables multi-omics analyses from one single CNS cell suspension and, eventually, the exploration of complex neuronal networks. Even if it is not a must to pool several tissues from multiple animals to perform this protocol, this pooling ensures an adequate number of isolated cells for further downstream analysis. The usage of different mice for the isolation of the single cell types would exclude the possibility of analyzing potential cellular interactions. Besides that, combining individual isolation methods for the different CNS cell types, which all follow a prior CNS dissociation, saves material costs by using one dissociated CNS cell suspension for all following magnetic isolation steps. Additionally, a potential technical bias caused by the usage of different mice is minimized.
One limitation of the protocol could be the nearly exclusive usage of female C57BL/6J mice. The EAE immunization protocol has been designed and established for female mice, so this cell isolation protocol was implemented in female C57BL/6J mice as well. Nevertheless, na&#239;ve male mice were also used during the development of this protocol, without recognizing any effect on the resulting cell number or purities. Another restriction affects the magnetic cell isolation of neurons as there exist no specific micro-beads for the isolation of neurons in terms of a positive selection. It was assumed that a pure single-cell suspension could be obtained via biotin labeling and depletion of all non-neuronal cells (Table 2). This assumption was verified by the usage of NeuN as a specific nuclear marker for neurons, integrated in the mentioned flow cytometry purity panel. Another limitation concerns the isolation of microglia in EAE mice. Here, resulting cell yields are decreased in comparison to the other cell types because of the additional sorting step after the MACS protocol. Moreover, one could argue that sorting increases the mechanical stress of the microglia compared to the other cell populations. Individual sorting strategies may lead to different amounts of cell yields. If isolated cell number is less than expected or desired, adjusting the gating set up and/or improving the live/dead discrimination is recommended.
A critical step in the protocol represents the debris removal. The gradient must be layered very slowly and gently to create the three wanted separate phases (Figure 2A). Only if the myelin and other debris residues in the two top phases are removed totally (Figure 2E), pure single-cell suspensions can be generated, and further contamination can be reduced. If the resulting cell suspensions lack purity, this is probably the section of the protocol that should be improved first next to the assurance of the right usage of all micro-beads.
Receiving high levels in purity and viability can be challenging in this type of experiment. Some recommendations for troubleshooting are:
-Working under sterile conditions is obligatory to prevent contamination of the different micro-beads and enable repeated use, especially for subsequent cultivation. 
-Labeling of each tube to prevent mix-ups is highly recommended. 
-Avoid the usage of uncooled reagents/buffers. Store all cell suspensions on ice during the whole experiment to ensure high viability. 
-Keep the time between the different working steps as short as possible. There is no specific part in the protocol where pausing the experiment is recommended. 
-It is highly relevant to adhere to the specified incubation periods.
In conclusion, this current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and neuroinflammatory pathways ex vivo from one CNS cell suspension. Thus, CNS-resident cells can be investigated during different stages of disease courses, e.g., during neuroinflammation, neurodegeneration and/or remission in EAE. Furthermore, cell-cell interactions and biochemical pathways can be studied on an individual level and variability within experimental groups can be reduced. There is also the opportunity to cultivate fractions of the isolated CNS cells in monocultures for further functional assays and validation. All in one, this protocol offers significant advances potentially affecting preclinical and clinical research approaches.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage, gliosis, and neurodegeneration. Despite numerous research approaches in this field, the pathophysiology of MS is still not fully understood1,2,3,4. The most common animal model for investigating MS is the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) which shares many of its clinical and pathophysiological features5,6,7,8,9. It is based on the response of the immune system against CNS-specific antigens leading to inflammation, demyelination, and neuroaxonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is a suitable model for the investigation of neuroinflammatory pathways and signaling cascades found in MS.
Current therapy options for MS are only partially effective and focus primarily on the initial inflammatory phase of the disease. However, the neurodegenerative component of MS seems to be the major challenge for long-term therapeutic approaches. Therefore, reproducible, and precise cell isolation protocols are required to investigate molecular and cellular mechanisms in autoimmune diseases in a comprehensive way. Even if some protocols for the isolation of one single cell type exist10,11,12,13,14,15, there is an unmet need for the simultaneous isolation of several CNS-resident cell populations at once. Previous protocols for the isolation of CNS-resident cells lack in preserving cellular functionality and purity, resulting in co-cultivation with neighboring cells16,17,18 or the unsuitability for complex analyses of intracellular networks ex vivo19,20,21,22.
The aim of this protocol was to establish a reproducible and comprehensive method for the simultaneous isolation of pure viable single-cell suspensions of all principal CNS-resident cell types applicable in adult healthy and EAE mice. The different cell types were isolated using magnetic-activated cell sorting (MACS)23. The cell separation can be accomplished either by positive selection, i.e., magnetic labeling of cell-type-specific surface markers, or by negative selection via biotinylation and depletion of all undesired cells. Flow cytometry was applied to ensure a purity of above 90% and a viability of at least 80% of the isolated single-cell suspensions.
In conclusion, the main goal was to establish a protocol for the simultaneous isolation of all main CNS-resident cell types as a versatile tool for the investigation of neuroinflammatory pathways offering a comprehensive and precise analysis of complex cellular networks and biochemical signaling cascades in healthy and EAE mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70 &#956;m cell strainers</td>
			<td>Corning, MA, USA</td>
			<td>352350</td>
			<td>CNS tissue dissociation</td>
		</tr>
		<tr>
			<td>ACSA-2 Antibody, anti-mouse, PE-Vio 615 (clone REA-969)</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-116-244</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit, mouse, and rat</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-107-677</td>
			<td>Tissue dissociation,contains debris and red blood cell removal solutions; prepare aliquots of enzyme A and P upon arrival and store them at -20 &#176;C; store the remaining kit at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-ACSA-2 MicroBead Kit, mouse</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-097-678</td>
			<td>MACS of astrocytes, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32 antibody&#160;</td>
			<td>BioLegend, London, UK</td>
			<td>101301</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-O4 MicroBeads, human, mouse, rat</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-094-543</td>
			<td>MACS of oligodendrocytes, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>AstroMACS Separation buffer</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-091-221</td>
			<td>MACS of astrocytes, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Biotin Antibody, PE (clone Bio3-18E7)</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-113-853</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>BRAND Neubauer counting chamber</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>10195580</td>
			<td>Cell counting&#160;</td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-mouse CD45 Antibody (clone 30-F11)</td>
			<td>BioLegend, London, UK</td>
			<td>103137</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>CD11b MicroBeads, human, mouse</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-049-601</td>
			<td>MACS of microglia, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DNAse I, recombinant, Rnase-free</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>4716728001</td>
			<td>Flow cytometry, store at -20&#176; C</td>
		</tr>
		<tr>
			<td>D-PBS with Calcium, Magnesium, Glucose, Pyruvat</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>14287080</td>
			<td>Buffer, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>D-PBS, without calcium, without magnesium</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>14190250</td>
			<td>Buffer, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>eBioscience Fixable Viability Dye eFluor 780</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>65-0865-14</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>eBioscience Foxp3/Transcription factor staining buffer set</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>00-5523-00</td>
			<td>Flow cytometry, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Falcon (15 mL)</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>11507411</td>
			<td>Cell tube</td>
		</tr>
		<tr>
			<td>Falcon (50 mL)</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>10788561</td>
			<td>Cell tube&#160;</td>
		</tr>
		<tr>
			<td>Falcon&#160;Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>08-771-23</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent, mouse&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-092-575</td>
			<td>MACS of oligodendrocytes, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Female C57BL/6J mice</td>
			<td>Charles River Laboratories, Sulzfeld, Germany</td>
			<td></td>
			<td>Active EAE induction&#160;</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>F2442-50ML</td>
			<td>Flow cytometry, store at -5 to -20 &#176;C</td>
		</tr>
		<tr>
			<td>FITC Rat Anti-CD 11b&#160; (clone M1/70)</td>
			<td>BD Biosciences, San Jose, CA, USA</td>
			<td>553310</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Freund&#8217;s Complete adjuvant</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>AR001</td>
			<td>Active EAE induction, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>GentleMACS C Tubes</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-093-237</td>
			<td>CNS tissue dissociation</td>
		</tr>
		<tr>
			<td>GentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-096-427</td>
			<td>CNS tissue dissociation</td>
		</tr>
		<tr>
			<td>Graphpad Prism 8.4.3</td>
			<td>Graphpad by Dotmatics</td>
			<td></td>
			<td>Graphical Analysis</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>AbbVie, North Chicago, IL, USA</td>
			<td></td>
			<td>Active EAE induction, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software V2.1.1</td>
			<td>Beckman Coulter, Indianapolis, IN, USA</td>
			<td></td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-042-401</td>
			<td>MACS</td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-091-376</td>
			<td>PB-buffer</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-042-303</td>
			<td>MACS&#160;</td>
		</tr>
		<tr>
			<td>MOG35&#8211;55 peptide</td>
			<td>Charit&#233;, Berlin, Germany; alternatives: Genosphere Biotechnologies (Paris, France) or sb-Peptide (Saint Egr&#232;ve, France)</td>
			<td></td>
			<td>Active EAE induction, store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis strain H37 Ra</td>
			<td>Becton, Dickinson and Company (BD),Franklin Lakes, NJ, USA</td>
			<td></td>
			<td>Active EAE induction, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neuron Isolation Kit, mouse&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-115-390</td>
			<td>MACS of neurons, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>O4 Antibody, anti-human/mouse/rat, APC, (clone REA-576)</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-119-897</td>
			<td>Flow cytometry, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Pertussis toxin in glycerol</td>
			<td>Hooke Laboratories Inc., Lawrence, MA, USA</td>
			<td>BT-0105</td>
			<td>Active EAE induction; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pluriStrainer Mini 100 &#956;m&#160;</td>
			<td>pluriSelect Life Science UG, Leipzig, Sachsen, Germany</td>
			<td>43-10100-40</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>QuadroMACS Separator&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td>
			<td>130-090-976</td>
			<td>MACS</td>
		</tr>
		<tr>
			<td>Recombinant Alexa Fluor 647 Anti-NeuN antibody (clone EPR12763)</td>
			<td>Abcam, Cambridge, UK</td>
			<td>EPR12763</td>
			<td>Flow cytometry, store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Stainless Steel Brain Matrices, 1 mm</td>
			<td>Ted Pella, Redding, CA, USA</td>
			<td>15067</td>
			<td>CNS tissue dissection</td>
		</tr>
		<tr>
			<td>Trypan blue solution, 0.4%</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>15250061</td>
			<td>Cell counting&#160;</td>
		</tr>
		<tr>
			<td>UltraPure 0.5&#160;M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific,Waltham, MA, USA</td>
			<td>15575020</td>
			<td>Flow cytometry, store at room temperature</td>
		</tr>
	</tbody>
</table>

simultaneous isolation of principal central nervous system-resident cell types from adult autoimmune encephalomyelitis mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and new inflammatory pathways, ex vivo from one single cell suspension, applicable to healthy mice and those with experimental autoimmune encephalomyelitis.Additionally, mice numbers are decreased Our protocol for the simultaneous isolation of all four major CNS-resident cell types in healthy and EAE mice could be used as a helpful tool for research groups studying new inflammatory pathways, allowing a much more accurate analysis of complex biomolecular mechanisms and cellular networks ex vivo. New scientific questions that could be answered with the help of our protocol are dynamic investigations in EAE during different stages of disease course, like neuroinflammation, neurodegeneration, and remission. Also, cell-cell interactions and biochemical pathways could be studied on an individual level.Likewise, functional assays could be performed with cultivated cells. We intend to focus on dynamic studies of the EAE course for multi-omic analysis from one individual CNS homogenate per EAE time point.

The current protocol offers the possibility to simultaneously isolate all principal CNS-resident cells, i.e., microglia, oligodendrocytes, astrocytes, and neurons from one single CNS replicate. This is important for the reduction of mice numbers needed for these kinds of experiments and to ensure the comparability of molecular and biochemical analyses on cellular level. If the individual cell types are isolated from different CNS replicates, cellular interactions cannot be mapped truthfully and potential technical deviations during the isolation processes could bias further downstream analyses. In addition, molecular and biochemical findings from each cell type would not be comparable to each other as they are not derived from the same EAE context. A pre-existing MACS protocol using a commercial system/kit was adapted to enable simultaneous isolation of the above-mentioned cell types.
The isolation of microglia was performed using anti-CD11b micro-beads, oligodendrocytes were isolated via anti-O4 micro-beads (Table 1), and anti-ACSA-2 micro-beads were used to isolate astrocytes (Table 2). In contrast, the isolation of neurons represents a negative selection and was accomplished by biotinylation and magnetic labeling of all non-neuronal cells (Table 2). All non-neuronal cells (e.g., oligodendrocytes, microglia, astrocytes, endothelial cells, and fibroblasts) except for blood cells can be magnetically labeled by using a biotin-conjugated antibody, specifically directed against a surface antigen expressed on these non-neuronal cells (Table 2). By depletion of these magnetically labeled non-neuronal cells, highly pure and viable neuronal cell populations can be generated30,42,43.
Two new flow cytometry panels for the purity analyses of the generated single-cell suspensions were designed. Here, cell-type-specific surface and nuclear markers combined with live/dead cell discrimination were used.
The resulting cell yields per mouse and cell-type (Figure 3A) were analyzed and led to an average of 8. 4 x 105 &#177; 3 x 104 microglia, 3.23 x 106 &#177; 1 x 105 oligodendrocytes, 8.6 x 105 &#177; 2 x 104 astrocytes, and 4.5 x 105 &#177; 1 x 104 neurons per na&#239;ve mouse.
In the context of the aim to investigate disease models of neuroinflammation, the protocol was also applied to a mouse model of EAE. The mice were euthanized on day 16 after EAE induction representing the disease maximum. In this EAE setting, approximately 2.9 x 106 &#177; 6.7 x 105 oligodendrocytes, 5.2 x 105 &#177; 9 x 104 astrocytes and 4.4 x 105 &#177; 4 x 104 neurons were isolated. The microglia cell yield was decreased to approximately 1.7 x 105 &#177; 4 x 104 microglia per EAE mouse because of the additional cell sorting after the MACS steps (Figure 3A).
After isolation, phenotypic characterizations of the different cell populations via flow cytometry proved that viable single-cell suspensions with a purity of approximately 90% for all main CNS-resident cell types (Figure 3B) could be achieved. Microglia were gated as being CD45intCD11bhigh as defined in the literature44,45,46,47.
In EAE, microglia had to be sorted from all CD11b+ cells to differentiate them from other CD11b+ immune cells like monocytes, neutrophils, natural killer cells, granulocytes, and macrophages that immigrate into the CNS during neuroinflammation27,28,48. Therefore, the microglia were sorted as CD45intCD11high cells from the CD11b+ cell suspension. The whole microglia sorting strategy is depicted in Figure 4. In na&#239;ve mice, the microglia population was 91.16% of all live single cells (96% of the total CD11b+ population) (Figure 4A). In EAE mice, the microglia population was 46.85% of all live single cells (55% of the total CD11b+ population) (Figure 4B). Although both MACS and FACS procedures apply mechanical stress to the single cells, 75.41% &#177; 8.63% of the sorted purified microglia were viable (Figure 3B).
Astrocytes and neurons that were directly isolated from the initial CNS cell suspension showed relevant contamination with oligodendrocytes which led to the assumption that the simultaneous isolation of neurons and astrocytes from the negative flow-through of oligodendrocytes could prevent this contamination. Flow cytometry analyses confirmed that astrocytes isolated from the negative flow-through of oligodendrocytes had a purity of 89.23% &#177; 2.78% and demonstrated a viability of 80.56% &#177; 1.41%. Similar to these results, the purity of the neurons isolated from the O4- cell fraction was 81.25% &#177; 3.31% and the viability was 83.90% &#177; 2.61% (Figure 3B). These findings also confirm that the simultaneous isolation of these two cell types only subsequently to the isolation of oligodendrocytes has no impact on the amount of viable functional cells.
The results regarding the viability and purity of the isolated single-cell suspensions were very similar in EAE mice compared to those received in na&#239;ve mice confirming that this protocol is suitable for healthy mice as well as in the context of EAE (Figure 3B).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65735/65735fig03.jpg" /> 
Figure 3: Cell yields and flow cytometry-based validation of isolated CNS-resident cells. (A) Cell yields per mouse and cell-type after isolation of CNS-resident cells in na&#239;ve and EAE mice. Bar graphs visualize the amount of cell yields per mouse and cell-type after the implementation of the presented protocol. Five biological replicates were processed for the results in na&#239;ve mice, and four biological replicates were analyzed in EAE mice. Respective means &#177; SEMs are depicted. (B) Corresponding purity and viability analyses of the purified cell fractions. Bar graphs indicate the viability and purity of the resulting single-cell suspensions based on their expression of cell-type-specific markers. NeuN was used as a cell-type specific nuclear marker for neurons. Five biological replicates were acquired and compared for each cell type for both healthy and EAE mice. Respective means &#177; SEMs are indicated.&#160;Abbreviations: Anti-ACSA-2 = astrocyte cell surface antigen-2; CD11b = cyclin-dependent kinase 11B; CD45 = receptor-type tyrosine-protein phosphatase C; CNS = central nervous system; EAE = experimental autoimmune encephalomyelitis; MACS = magnetic-activated cell sorting; NeuN =RNA binding protein fox-1 homolog 3; O4 = oligodendrocyte marker O4; SEM = standard error of the mean. This figure has been modified from49. <a href="https://www.jove.com/files/ftp_upload/65735/65735fig03large.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/65735/65735fig04.jpg" /> 
Figure 4: Gating strategy for cell sorting of microglia after isolation of CD11b+ cells. (A) Gating strategy in na&#239;ve and (B) EAE mice. The upper row of each panel shows dot plots before sorting and the lower row after sorting. After selection of live (SSC-A / FSC-A) and single cells (FCS-H / FSC-W), the population of CD45intCD11b+ cells were sorted as microglia population. <a href="https://www.jove.com/files/ftp_upload/65735/65735fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types at JoVE.com

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

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JoVE Journal

Neuroscience

806 Views.  Heinrich Heine University Duesseldorf. To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to ana...

Video: Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

Preparation of CNS Tissue for Isolating Cells in EAE Mice 

Begin by placing the mouse in supine position and fixing the limbs with needles. Apply 75%ethanol on the front body of the animal. Make a longitudinal section through the skin and fascia on the abdomen and thorax.Cut the ribs parasternal and fold up the thorax to gain free access to the heart. Fix the thorax folded upwards with needles. Open the right atrium using scissors.Using a cannula, apply 20 milliliters of one-time PBS into the left ventricle to flush out the blood through the incised right atrium. Make a longitudinal section by cutting the skin on top of the head of the mouse to expose the skull and shift the skin around the head using forceps. Incise the skull with the help of a scissor along the sagittal suture.Insert the tip of the scissors along the incision line to crack open the calotte. Remove the remaining parts of the calotte with forceps so that the brain is fully exposed. Remove the brain carefully and place it into a murine brain matrix.Cut the brain into one millimeter thick sagittal slices by using a razor blade. With the help of scissors, cut the vertebral column just above the iliac crest. Insert a 20 gauge needle attached to a 20 milliliter syringe containing one-time PBS into the spinal canal.Flush the spinal cord out of the spinal canal from caudal to cranial. Cut the spinal cord into 0.5 centimeter long segments using a scalpel. Store the CNS cell suspension consisting of the brain and corresponding spinal cord in a Petri dish containing three milliliters of cold DPBS.

Dissociation of CNS Tissue for Isolating Cells in EAE Mice 

To begin, dissect the brain and spinal cord tissue and store the cell suspension on ice. Then prepare an appropriate volume of enzyme mix-1 and 2. Transfer 1, 950 microliters of enzyme mix-1 into the C tube, and add the tissue pieces of the CNS cell suspension.Then add 30 microliters of enzyme mix-2 to the C-tube. Close the C-tubes tightly and attach them upside down onto the sleeve of the cell dissociator with heaters. Run the appropriate program and observe for at least five minutes to ensure all tubes turn at the same velocity.In the meantime, place a 70-micron strainer on a 50-milliliter tube and pre-moisten the strainer with two milliliters of DPBS. After termination of the program, detach the C tubes from the dissociator and centrifuge the samples at 300 g for one minute at four degrees Celsius. Resuspend the sample and apply it to the pre-moistened strainer.Add 10 milliliters of cold DPBS to the empty C-tube. Mix and add the suspension onto the corresponding strainer. After discarding the strainers, centrifuge the cell suspension again for 10 minutes, and then carefully aspirate the supernatant to obtain a cell pellet.

Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice

To begin, obtain a cell pellet after dissociation of CNS tissue and re-suspend it with 3, 100 microliters of DPBS in one 15-milliliter tube. Do not vortex. Add 1, 800 microliters of the debris removal solution from the adult brain dissociation kit to two pooled CNS cell suspensions.Invert the tube and mix the suspension, then gently overlay it with 4 milliliters of cold DPBS and observe a clear gradient. Centrifuge the tubes for 10 minutes at 3, 000g at 4 degrees Celsius with full acceleration and no break. After centrifugation, three phases are formed.Aspirate the two top phases completely and discard them, ensuring no myelin residues are left behind. Fill up the tube with cold DPBS up to 14 milliliters and close it. Invert the tube with force on the workbench until the cell pellet becomes detached from the bottom of the tube.Centrifuge the sample and aspirate the supernatant completely. For red blood cell removal, add 1 milliliter of the red blood cell removal solution to the cell pellet. After re-suspending the pellet, incubate the solution for 10 minutes at 4 degrees Celsius.Then add 10 milliliters of cold PB buffer, centrifuge the sample, and completely aspirate the supernatant. For cell counting, dilute the cell suspension by 50 times in PB buffer and then by 1 to 10 dilution in 0.4%Trypan Blue solution. Then determine the cell count, using an improved counting chamber.

Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice

To begin, divide the purified, undiluted mouse CNS cell suspension into two fractions for further isolations of microglia and oligodendrocytes. Then add magnetically labeled microbead specific for the surface antigen for different CNS cell types to the respective cell suspension. Place the column in the magnetic field, equate the column, and then add the resulting cell suspension onto the column.Magnetically labeled cells will be retained within the column, and unlabeled cells will run through. After removing the column from the magnetic field, flush out magnetically labeled cells from the column into a tube as the positively selected cell fraction. Divide the negative flow through from the oligodendrocytes into two fractions for the further isolations of neurons and astrocytes.Flow cytometry analysis using cell type specific markers combined with live or dead cell discrimination yielded microglia, oligodendrocytes, astrocytes, and neurons. Phenotypic characterization of the different cell populations proved that single cell suspensions with approximately 90%purity and viability were obtained for all main CNS resident cell types.