人物是使用 Adobe Illustrator（2023 版）和 Servier Medical Art （https://smart.servier.com）创建的。安东尼娅·赫内斯（Antonia Henes）得到了于尔根·曼肖基金会（Jürgen Manchot Stiftung）的支持。

从自身免疫小鼠中同时分离所有 CNS 驻留细胞类型

所有作者均声明没有利益冲突。

到目前为止，通过结合质谱法和 RNA 测序来绘制 CNS 驻留细胞离体的方法在健康和疾病中提供了非常精确的细胞谱分析，但需要该领域雄心勃勃的技术知识和专业知识50,51。此外，它们不允许进行泛函分析，并且非常昂贵。除此之外，微流控脑芯片系统为疾病机制提供了快速且经济实惠的筛选，并测试了限制细胞生长和迁移的新治疗方法52,53,54,55。CNS类器官也可能在未来代表一种等效的替代方案，用于研究疾病过程中的细胞建模、细胞间连接和相互作用56,57,58,59。然而，荧光和磁激活细胞分选是目前产生纯和活单细胞悬浮液的最有效方法35,60,61。即使其他已建立的用于分离 CNS 驻留细胞类型的制造方案在磁隔离和先前细胞解离的各个步骤方面相似，它们也旨在针对每种细胞类型单独执行。相比之下，目前的方案将每种 CNS 驻留细胞类型的不同分离方法集成到逻辑上下文中，以便它们可以同时从一个单个 CNS 细胞悬液中执行（表 1、表 2）。因此，它能够从单个CNS细胞悬液进行多组学分析，并最终探索复杂的神经元网络。即使不需要混合来自多个动物的多个组织来执行该方案，这种合并也确保了足够数量的分离细胞用于进一步的下游分析。使用不同的小鼠来分离单细胞类型将排除分析潜在细胞相互作用的可能性。除此之外，将不同中枢神经系统细胞类型的单独分离方法（均遵循先前的中枢神经系统解离）相结合，通过将一种解离的中枢神经系统细胞悬浮液用于所有后续磁性分离步骤，可以节省材料成本。此外，由于使用不同的鼠标而引起的潜在技术偏差被最小化。
该协议的一个局限性可能是几乎完全使用雌性C57BL / 6J小鼠。EAE免疫方案是针对雌性小鼠设计和建立的，因此该细胞分离方案也在雌性C57BL / 6J小鼠中实施。然而，在该方案的开发过程中也使用了幼稚的雄性小鼠，没有认识到对所得细胞数量或纯度的任何影响。另一个限制会影响神经元的磁性细胞分离，因为在正选择方面不存在用于分离神经元的特定微珠。假设可以通过生物素标记和所有非神经元细胞的耗竭获得纯单细胞悬浮液（表2）。通过使用 NeuN 作为神经元的特异性核标记物来验证这一假设，该标记物集成在上述流式细胞术纯度面板中。另一个局限性涉及EAE小鼠中小胶质细胞的分离。在这里，与其他细胞类型相比，由于MACS协议之后的额外分选步骤，产生的细胞产量降低。此外，有人可能会争辩说，与其他细胞群相比，分选增加了小胶质细胞的机械应力。单独的分选策略可能导致不同数量的细胞产量。如果分离的细胞数量小于预期或期望，建议调整门控设置和/或改进活/死区分。
协议中的一个关键步骤是清除碎片。梯度必须非常缓慢而温和地分层，以创建三个想要的独立相（图2A）。只有当两个顶相中的髓鞘和其他碎片残留物被完全去除（图2E），才能产生纯单细胞悬浮液，并减少进一步的污染。如果得到的细胞悬浮液缺乏纯度，这可能是方案中应该首先改进的部分，其次是保证所有微珠的正确使用。
在这种类型的实验中，获得高水平的纯度和活力可能具有挑战性。一些故障排除建议包括：
-必须在无菌条件下工作，以防止不同微珠的污染，并允许重复使用，特别是用于后续培养。 -强烈建议在每根管子上贴上标签以防止混淆。 -避免使用未冷却的试剂/缓冲液。在整个实验过程中，将所有细胞悬浮液储存在冰上，以确保高活力。 -保持不同工作步骤之间的时间尽可能短。协议中没有建议暂停实验的特定部分。 -遵守规定的潜伏期非常重要。
总之，目前从一次中枢神经系统复制中同时分离所有主要中枢神经系统驻留细胞类型的方案提供了从一次中枢神经系统细胞悬浮液中分析复杂神经元网络和神经炎症途径 的可能性 。因此，可以在病程的不同阶段研究驻留中枢神经系统细胞，例如，在 EAE 的神经炎症、神经退行性和/或缓解期间。此外，可以在个体水平上研究细胞间相互作用和生化途径，并且可以减少实验组内的变异性。还有机会在单一培养物中培养分离的中枢神经系统细胞的组分，以进行进一步的功能测定和验证。总而言之，该协议提供了可能影响临床前和临床研究方法的重大进展。

多发性硬化症 （MS） 是一种中枢神经系统 （CNS） 的慢性炎症性自身免疫性疾病，其特征是脱髓鞘、轴突损伤、神经胶质增生和神经退行性变。尽管该领域有许多研究方法，但 MS 的病理生理学仍未完全了解 1,2,3,4。研究 MS 的最常见动物模型是髓鞘少突胶质细胞糖蛋白肽 35-55 （MOG35-55） 诱导的实验性自身免疫性脑脊髓炎 （EAE），它具有许多临床和病理生理特征 5,6,7,8,9 .它基于免疫系统对导致炎症、脱髓鞘和神经轴突变性的中枢神经系统特异性抗原的反应。实验性自身免疫性脑脊髓炎 （EAE） 是研究 MS 中发现的神经炎症通路和信号级联反应的合适模型。
目前多发性硬化症的治疗方案仅部分有效，主要集中在疾病的初始炎症阶段。然而，多发性硬化症的神经退行性成分似乎是长期治疗方法的主要挑战。因此，需要可重复和精确的细胞分离方案来全面研究自身免疫性疾病的分子和细胞机制。即使存在一些用于分离一种单一细胞类型的方案10,11,12,13,14,15，同时分离多个CNS驻留细胞群的需求仍未得到满足。先前用于分离中枢神经系统驻留细胞的方案缺乏保留细胞功能和纯度的方案，导致与相邻细胞共培养16,17,18或不适合离体细胞内网络的复杂分析19,20,21,22。
该方案的目的是建立一种可重复和全面的方法，用于同时分离适用于成年健康和EAE小鼠的所有主要CNS驻留细胞类型的纯活单细胞悬浮液。使用磁活化细胞分选 （MACS） 23 分离不同的细胞类型。细胞分离可以通过阳性选择（即细胞类型特异性表面标记物的磁性标记）或通过生物素化和所有不需要的细胞的耗竭进行阴性选择来实现。应用流式细胞术以确保纯度高于 90% 和至少 80% 的分离单细胞悬浮液的活力。
总之，主要目标是建立一种同时分离所有主要中枢神经系统驻留细胞类型的方案，作为研究神经炎症途径的多功能工具，提供对复杂细胞网络和生化信号级联的全面精确分析健康和EAE小鼠。

<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

实验性自身免疫性脑脊髓炎 （EAE） 是多发性硬化症 （MS） 最常见的小鼠模型，经常用于进一步阐明 MS 的未知病因，以开发新的治疗策略。髓鞘少突胶质细胞糖蛋白肽 35-55 （MOG35-55） EAE 模型再现免疫后 10 天内伴有上行麻痹的自限性单相病程。每天使用临床评分系统检查小鼠。多发性硬化症由具有特定时间模式的不同病理机制驱动，因此研究中枢神经系统 （CNS） 驻留细胞类型在疾病进展过程中的作用非常有意义。该协议的独特之处在于同时分离适用于成年EAE和健康小鼠的所有主要CNS驻留细胞类型（小胶质细胞，少突胶质细胞，星形胶质细胞和神经元）。从成年小鼠中分离大脑和脊髓后，进行磁活化细胞分选 （MACS） 以分离小胶质细胞、少突胶质细胞、星形胶质细胞和神经元。流式细胞术用于对纯化的单细胞悬液进行质量分析，确认细胞分离后的活力，并表明每种细胞类型的纯度约为 90%。总之，该协议提供了一种精确而全面的方法来分析健康和EAE小鼠中的复杂细胞网络。此外，由于所有四种细胞类型都是从同一只小鼠中分离出来的，因此所需的小鼠数量可以大大减少。

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.

作者聚焦：通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

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.

目前的方案提供了从单个中枢神经系统复制中同时分离所有主要中枢神经系统驻留细胞的可能性，即小胶质细胞、少突胶质细胞、星形胶质细胞和神经元。这对于减少此类实验所需的小鼠数量以及确保细胞水平上分子和生化分析的可比性非常重要。如果从不同的中枢神经系统重复中分离出单个细胞类型，则无法如实绘制细胞相互作用图谱，并且分离过程中潜在的技术偏差可能会进一步偏向下游分析。此外，每种细胞类型的分子和生化发现不会相互比较，因为它们不是来自相同的EAE背景。使用商业系统/试剂盒的预先存在的 MACS 方案经过调整，以实现上述细胞类型的同时分离。
使用抗CD11b微珠分离小胶质细胞，通过抗O4微珠分离少突胶质细胞（表1），使用抗ACSA-2微珠分离星形胶质细胞（表2）。相比之下，神经元的分离代表了一种负选择，并且是通过所有非神经元细胞的生物素化和磁性标记完成的（表2）。除血细胞外，所有非神经元细胞（例如少突胶质细胞、小胶质细胞、星形胶质细胞、内皮细胞和成纤维细胞）都可以通过使用生物素偶联抗体进行磁性标记，特异性针对这些非神经元细胞上表达的表面抗原（表 2）。通过耗尽这些磁性标记的非神经元细胞，可以产生高纯度和活的神经元细胞群30,42,43。
设计了两种新的流式细胞术panel，用于生成的单细胞悬液的纯度分析。在这里，使用了细胞类型特异性表面和核标记物以及活/死细胞区分。
分析了每只小鼠和细胞类型的细胞产量（图3A），平均为8。每只幼稚小鼠 4 x 105 ± 3 x 104 个 小胶质细胞、3.23 x 106 ± 1 x 105 个 少突胶质细胞、8.6 x 105 ± 2 x 104 个 星形胶质细胞和 4.5 x 105 ± 1 x 104 个神经元。
在旨在研究神经炎症疾病模型的背景下，该协议还应用于EAE的小鼠模型。在EAE诱导后的第16天对小鼠实施安乐死，代表疾病最大值。在这种 EAE 环境中，分离了大约 2.9 x 106 ± 6.7 x 105 个少突胶质细胞、5.2 x 105 ± 9 x 104 个星形胶质细胞和 4.4 x 105 ± 4 x 104 个神经元。由于MACS步骤后的额外细胞分选，小胶质细胞产量降低至每只EAE小鼠约1.7×105±4×10.4小胶质细胞（图3A）。
分离后，通过流式细胞术对不同细胞群进行表型表征证明，对于所有主要的中枢神经系统驻留细胞类型，可以实现纯度约为 90% 的活单细胞悬液（图 3B）。小胶质细胞被门控为CD45intCD11b高，如文献44,45,46,47所定义。
在 EAE 中，必须从所有 CD11b+ 细胞中分选小胶质细胞，以将它们与其他 CD11b+ 免疫细胞（如单核细胞、中性粒细胞、自然杀伤细胞、粒细胞和巨噬细胞）区分开来，这些细胞在神经炎症期间迁移到中枢神经系统 27,28,48。因此，小胶质细胞从CD11b+细胞悬液中分选为CD45intCD11高细胞。整个小胶质细胞分选策略如图4所示。在幼稚小鼠中，小胶质细胞群占所有活单细胞的91.16%（占CD11b +总数的96%）（图4A）。在EAE小鼠中，小胶质细胞群体占所有活单细胞的46.85%（占CD11b +总数的55%）（图4B）。尽管MACS和FACS程序都对单细胞施加机械应力，但75.41%±8.63%的分选纯化小胶质细胞是可行的（图3B）。
从初始中枢神经系统细胞悬液中直接分离的星形胶质细胞和神经元显示出少突胶质细胞的相关污染，这导致假设神经元和星形胶质细胞同时从少突胶质细胞的负流中分离可以防止这种污染。流式细胞术分析证实，从少突胶质细胞负流过中分离出的星形胶质细胞纯度分别为89.23%±2.78%，活力分别为80.56%±1.41%。与这些结果类似，从O4细胞 部分分离的神经元纯度为81.25%±3.31%，活力分别为83.90%±2.61%（图3B）。这些发现还证实，仅在分离少突胶质细胞之后同时分离这两种细胞类型对活功能细胞的数量没有影响。
与幼稚小鼠相比，EAE小鼠中分离的单细胞悬浮液的活力和纯度的结果非常相似，这证实了该方案适用于健康小鼠以及EAE的背景（图3B）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65735/65735fig03.jpg"> 图 3：分离的 CNS 驻留细胞的细胞产量和基于流式细胞术的验证。（A） 在幼稚小鼠和 EAE 小鼠中分离 CNS 驻留细胞后每只小鼠和细胞类型的细胞产量。条形图可视化了实施所提出的方案后每只小鼠和细胞类型的细胞产量。在幼稚小鼠中处理了 5 个生物学重复的结果，并在 EAE 小鼠中分析了 4 个生物学重复。描述了SEMs±各自的手段。（B） 纯化细胞组分的相应纯度和活力分析。条形图根据其细胞类型特异性标记物的表达来指示所得单细胞悬液的活力和纯度。NeuN 被用作神经元的细胞类型特异性核标志物。采集了五个生物学重复，并比较了健康和EAE小鼠每种细胞类型的生物学重复。标明了SEM±各自的手段。缩写：抗ACSA-2=星形胶质细胞表面抗原-2;CD11b = 细胞周期蛋白依赖性激酶 11B;CD45 = 受体型酪氨酸蛋白磷酸酶 C;CNS = 中枢神经系统;EAE = 实验性自身免疫性脑脊髓炎;MACS = 磁激活细胞分选;NeuN =RNA结合蛋白fox-1同源物3;O4 = 少突胶质细胞标志物 O4;SEM = 平均值的标准误差。这个数字是从49修改过来的。 <a href="https://www.jove.com/files/ftp_upload/65735/65735fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65735/65735fig04.jpg"> 图 4：分离 CD11b+ 细胞后小胶质细胞分选的门控策略。（A） 幼稚小鼠的门控策略和（B）EAE小鼠的门控策略。每个面板的上行显示排序前的点阵图，排序后的下行显示点阵图。选取活细胞（SSC-A/FSC-A）和单细胞（FCS-H/FSC-W）后，将CD45intCD11b+ 细胞群分类为小胶质细胞群。 <a href="https://www.jove.com/files/ftp_upload/65735/65735fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

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

迄今为止，从同一只小鼠中同时分离所有主要中枢神经系统驻留细胞类型的方案尚未满足需求。该方案显示了适用于幼稚和实验性自身免疫性脑脊髓炎小鼠的程序，以研究神经炎症期间的复杂细胞网络，同时减少所需的小鼠数量。

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

为了研究自身免疫性疾病（如多发性硬化症）的分子和细胞机制，生成可重复和复杂的细胞分离方案是一项未满足的需求。目前大多数用于分离和分析中枢神经系统驻留细胞的技术都显示出严重的缺点，例如仅关注一种细胞类型或仅关注出生后小鼠。目前从一次中枢神经系统复制中同时分离所有主要中枢神经系统驻留细胞类型的方案提供了分析复杂神经元网络和新的炎症途径的可能性，离体来自一个单细胞悬浮液，适用于健康小鼠和实验性自身免疫性脑脊髓炎患者。此外，小鼠数量减少 我们在健康和EAE小鼠中同时分离所有四种主要CNS驻留细胞类型的方案可以用作研究新炎症途径的研究小组的有用工具，从而可以更准确地分析复杂的生物分子机制和离体细胞网络。在我们的协议的帮助下可以回答的新科学问题是在疾病过程的不同阶段（如神经炎症、神经退行性变和缓解）对 EAE 的动态研究。此外，可以在个体水平上研究细胞间相互作用和生化途径。同样，可以用培养的细胞进行功能测定。我们打算专注于 EAE 课程的动态研究，用于从每个 EAE 时间点的单个 CNS 匀浆进行多组学分析。

simultaneous-isolation-principal-central-nervous-system-resident-cell

Research

JoVE Journal

Neuroscience

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

741 次观看.  Heinrich Heine University Duesseldorf. 为了研究自身免疫性疾病（如多发性硬化症）的分子和细胞机制，生成可重复和复杂的细胞分离方案是一项未满足的需求。目前大多数用于分离和分析中枢神经系统驻留细胞的技术都显示出严重的缺点，例如仅关注一种细胞类型或仅关注出生后小鼠。目前从一次中枢神经系统复制中同时分离所有主要中枢神经系统驻留细胞类型的方案提供了分析复杂神经元网络和新的炎症途?...

视频: 作者聚焦：通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

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.

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.

科研

神经科学

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.

用于分离EAE小鼠细胞的CNS组织的制备 

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.

用于分离EAE小鼠细胞的CNS组织的解离 

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.

从EAE小鼠的中枢神经系统细胞悬液中去除碎片和红细胞

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.