这项工作得到了国家科学技术委员会（CONACYT）（702361）授予的奖学金的支持。作者承认博士国立理工学院国立生物科学学院生物化学科学课程。

该研究是根据墨西哥官方标准NOM-062-ZOO-1999和实验动物护理和使用指南进行的。该研究获得了墨西哥儿童医院研究、伦理和生物安全委员会 （HIM/2023/006） 和墨西哥总医院研究和生物伦理委员会 Eduardo Liceaga 的批准 （DI/21/501/04/62）。从墨西哥儿童医院获得三只 6 至 8 周龄的 C57BL/6 小鼠，在隔离条件下在通风架中饲养，符合机构动物护理和使用指南。
1.材料和试剂的制备
<ol><li>将不含Ca2+ 和Mg2+ 的PBS以及Hank平衡盐溶液（HBSS）预热至37°C。</li>
	<li>用 100 μg/mL 的 Liberase 和 4 μg/mL 的 DNase （HBSS-LD） 补充 HBSS 溶液。确保溶液的生理pH值保持在7.4。 注意：每条脊髓需要 1 mL 的工作溶液。</li>
	<li>准备用于密度梯度离心的等渗90%溶液（市售，见 材料表）并将其预热至37°C。</li>
	<li>通过将5%热灭活胎牛血清（FBS）与PBS混合来制备染色缓冲液，并将混合物储存在冰上。</li>
	<li>将Dulbecco改良的Eagle's中高葡萄糖（1g / L）（DMEM）预热至37°C。</li>
	<li>用 10% FBS、2 mM L-谷氨酰胺、200 U/mL 青霉素、200 μg/mL 链霉素和 500 pg/mL 两性霉素 B（参见 材料表）补充 DMEM 培养基以制备 DMEM-S。</li>
</ol>2. 脊髓的解剖和准备
<ol><li>用腹膜内过量的戊巴比妥以150mg / kg的剂量对小鼠实施安乐死。使用70%乙醇对小鼠的胸部和背部进行消毒。 注意：在进行小鼠解剖之前，请确认安乐死成功。</li>
	<li>剃掉胸腰椎区域，并用胶带将四肢固定在解剖板上。</li>
	<li>使用手术刀，沿着从枕骨到骶骨区域的背中线小心切开（图2）。</li>
	<li>在立体手术显微镜的帮助下，分层解剖，直到到达腰椎并识别最后一个肋弓。 注意：13 个肋弓在胸椎中铰接;解剖时，它们变得比 L1 椎骨2 更上可见。</li>
	<li>确定最后一个颈椎和骶骨，然后切开脊柱（图2）。</li>
	<li>利用解剖钳，进行椎骨背侧椎板切除术以提取脊髓（图3A-D）。 注：除前两个颈椎（寰椎和轴椎）外，所有可移动椎骨在各个区域（颈椎、胸椎或腰椎）都具有共同的形态设计。每个典型的可活动椎骨在其前端都有一个圆柱形椎体。附着在身体后部的是称为椎弓（神经弓）的骨弓，由椎板和棘突组成。在这些结构之间是椎孔15。</li>
	<li>切除通过孔（背根和腹根）出现的神经，这些神经构成周围神经系统，可能含有浸润的巨噬细胞（图3D）。 注意：没有必要去除软脑膜或脑膜，因为密度梯度培养基可有效消除污染的星形胶质细胞。此外，保留脑膜可缩短纯化时间并增强活力。</li>
</ol>3.小胶质细胞的组织消化和分离
<ol><li>将脊髓放在解剖板上，使用 Vannas 剪刀将脊髓切成 2 毫米的小块，平均产生 10 块。</li>
	<li>将脊髓切片转移到2mL平底微管中（图3E，F）。</li>
	<li>加入 1 mL HBSS-LD 工作溶液（步骤 1.2）。在37°C孵育25分钟，每5分钟剧烈涡旋一次。</li>
	<li>将每个消化的内容物通过 40 μm 过滤器，然后加入 7 mL 含有 2% FBS 的 HBSS 以中和消化。</li>
	<li>使用 1 mL 注射器柱塞粉碎剩余的组织。将悬浮液在50mL锥形管中以220× g，4°C离心5分钟。</li>
	<li>使用 1 mL 微量移液管弃去上清液，并将沉淀重悬于 15 mL 锥形管中含有 2% FBS 的 2 mL HBSS 中。与 2 mL 90% 等渗密度梯度培养基混合。在160×g，18°C下离心25分钟。 注意： 利用缓慢加速并避免使用制动器。</li>
	<li>使用移液器取回接口并将其转移到 15 mL 锥形管中。用10mL PBS洗涤细胞，并在220× g，4°C下离心5分钟。</li>
	<li>将细胞重悬于DMEM-S（步骤1.6）中，并将它们储存在冰上。</li>
</ol>4. 确定纯度/活力
<ol><li>为了使用血细胞计数器室定量活细胞，使用0.4%台盼蓝染色剂（参见 材料表）。 注：平均而言，一只小鼠提供4-600万个细胞。</li>
	<li>要 通过 流式细胞仪定量活细胞，请使用市售的胺反应性荧光染料染色（参见 材料表）。<ol><li>将 100 万个细胞转移到 5 mL 聚苯乙烯圆底 （FACS） 管中。用PBS以1：1000的比例稀释荧光染料（产生活性工作溶液）。</li>
		<li>将样品与活力溶液在4°C的黑暗中孵育30分钟。用冰冷的PBS洗涤细胞两次。将细胞重悬于400μL染色缓冲液中（步骤1.4）。</li>
		<li>在4°C下用2.4G2抗FcRIII / I（参见 材料表）在冰冷的染色缓冲液中封闭细胞15分钟。</li>
		<li>通过将荧光标记的单克隆抗体 CD45-PerCP 和 CD11b-eFluor 450 加入染色缓冲液中（稀释度为 1：300）（参见 材料表）来配制抗体染色混合物。 注意：虽然这两种技术都可用，但请选择最符合后续协议的技术。</li>
		<li>将样品与抗体染色混合物在4°C的黑暗中孵育30分钟。用冰冷的染色缓冲液洗涤细胞两次。将细胞重悬于 400 μL 染色缓冲液中。</li>
		<li>在流式细胞仪上采集 250,000 个事件，如步骤 6 中的设门策略所述（图 4）。</li>
	</ol></li>
</ol>5.免疫荧光染色
<ol><li>将预处理的盖玻片放入 6 孔板的孔中。 注：要处理盖玻片，将它们置于含有500μL-聚赖氨酸（0.01mg / mL，参见 材料表）溶液的培养皿中，并孵育1小时。然后，用蒸馏水清洗三次，然后晾干。</li>
	<li>使用 500 μL DMEM-S 溶液将 200,000 个细胞接种到处理过的盖玻片上。孵育24小时。</li>
	<li>取出盖玻片，在室温下使用3.7%多聚甲醛（PFA）固定细胞20分钟。用冰冷的PBS清洗盖玻片三次。</li>
	<li>在室温下用0.2%Triton X-100在PBS中透化细胞15分钟。随后，用冷PBS洗涤盖玻片三次。在室温下用0.2%BSA在PBS中封闭细胞1小时。</li>
	<li>在封闭溶液中稀释荧光标记的单克隆抗体（以1：300稀释度，参见 材料表），并在室温下孵育1小时。用冰冷的PBS清洗盖玻片三次。</li>
	<li>将含有DAPI的封固剂涂在载玻片上，然后用盖玻片密封。 注意：载玻片可以立即使用荧光或共聚焦显微镜进行分析，也可以在 -20°C 下储存长达 3 个月。</li>
</ol>6. 脊髓小胶质细胞的门控策略
<ol><li>生成点图并建立门以基于活力染色和空荧光通道16 分离活细胞。排除非活细胞。</li>
	<li>利用前向和侧向散射参数生成另一个图以消除碎片16。</li>
	<li>开发额外的点图并分析 CD11b 和 CD45 的表达以区分小胶质细胞。</li>
</ol>

小鼠脊髓中小胶质细胞的提取和酶促纯化

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作者声明没有利益冲突。

由于小胶质细胞在大脑稳态中的重要性，已经开发了许多用于研究小胶质细胞的方案。在这些方法中，小胶质细胞通常来源于胚胎或新生大鼠和小鼠的大脑半球17。有限数量的研究涉及从成年小鼠脊髓中纯化小胶质细胞13,14。这些技术包括使用胶原酶和/或木瓜蛋白酶以及DNAse进行酶消化，通常与使用Percoll或OptiPrep的梯度分离相结合。然?...

脊椎动物的定义特征是脊柱或脊柱，其中脊索已被一系列称为椎骨的分段骨骼所取代，这些骨骼被椎间盘分割。这种骨质物质的连续性塑造了椎管，椎管是一个包围和保护脊髓的腔1。在啮齿动物属中，脊柱通常由7个颈椎、13个胸椎、6个腰椎和数量不等的尾椎2,3组成。脊髓的长度与脊柱的长度相似，末端丝是将脊髓固定在骶骨上的非神经结构。此外，神经纤维通过椎间孔1 排出。
哺乳动物中枢神经系统的发育和正常功能在很大程度上取决于神经系统常驻巨噬细胞（称为小胶质细胞4）的活动。尽管小胶质细胞最初被描述为大脑驻留吞噬细胞，但最近的研究将许多动态功能归因于这些细胞 5,6。小胶质细胞的大小在稳态中为 7 至 10 μm;它们被认为是体内用途最广泛的细胞之一，可以在形态和功能上适应不断变化的环境7.这些细胞在胚胎期和成体阶段都表现出高度异质性8,9，而在成体阶段，它们也表现出基于其时空背景的复杂功能异质性10。小胶质细胞的异质性和多种功能允许在脊髓和大脑中产生不同的基因表达和行为。研究表明，与大脑相比，CD11b、CD45、CD86 和 CCR9 在脊髓中的表达更高 8,9。
脑小胶质细胞分离存在多种方案11,12;然而，脊髓小胶质细胞只有少数存在13,14。优化从脊髓中纯化小胶质细胞的方法有助于开展多项专注于发现小胶质细胞生理学的研究。该协议旨在描述小鼠脊髓的简单且高度可重复的提取和小胶质细胞的纯化（图1）。

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	<tbody>
		<tr>
			<td>15 mL collection tubes</td>
			<td>Corning, USA</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microtubes</td>
			<td>Axygen, USA</td>
			<td>MCT-200-G</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2 anti-FcR</td>
			<td>BioLegend, USA</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL collection tubes</td>
			<td>Corning, USA</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b)</td>
			<td>Gibco, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody CD11b eFluor 450 anti-mouse</td>
			<td>eBioscience, USA</td>
			<td>48-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody CD45 PerCP anti-mouse&#160;&#160;</td>
			<td>BioLegend, USA</td>
			<td>103130</td>
			<td></td>
		</tr>
		<tr>
			<td>Balanced salt solution (PBS) calcium- magnesium-free</td>
			<td>Corning, USA</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Cell Strainer 40 &#956;m</td>
			<td>Corning, USA</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well Clear Not Treated&#160;</td>
			<td>Corning, USA</td>
			<td>CLS3736</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Heating Shaking Drybath&#160;</td>
			<td>Thermo Scientific Digital HS Drybath, USA</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps for microsurgery</td>
			<td>FT by DUMONT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche, USA</td>
			<td>4536282001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle&#180;s Medium-high glucose (DMEM)&#160;</td>
			<td>Merck, USA</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Thermo, USA</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAN Biotech, Alemania</td>
			<td>P30-3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer Cytoflex&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution&#160;</td>
			<td>Merck, USA</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning, USA&#160;</td>
			<td>15393631</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM&#160;</td>
			<td>Roche, USA</td>
			<td>5401119001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber Counting Chambers</td>
			<td>China</td>
			<td>1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>Merck, USA</td>
			<td>17089101</td>
			<td>density gradient centrifugation&#160;</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution&#160;</td>
			<td>Merck, USA</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel No. 25&#160;</td>
			<td>HERGOM, Mexico</td>
			<td>H23</td>
			<td></td>
		</tr>
		<tr>
			<td>Snaplock Microcentrifuge Tubes 2 mL</td>
			<td>Axygen, USA</td>
			<td>10011-680</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope</td>
			<td>Velab, Mexico</td>
			<td>HG927831</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight surgical scissors (10 cm)</td>
			<td>HERGOM, Mexico</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Vannas scissors</td>
			<td>HERGOM, Mexico</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Merck, USA</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain 0.4%&#160;</td>
			<td>Merck, USA</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>DLAB, China</td>
			<td>8031102000</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Kit</td>
			<td>BioLegend, USA</td>
			<td>423102</td>
			<td>amine-reactive fluorescent dye staining&#160;</td>
		</tr>
	</tbody>
</table>

rapid and efficient enrichment of mouse spinal cord microglia

脊柱定义了脊椎动物并塑造了椎管，椎管是一个包围和保护脊髓的腔体。哺乳动物中枢神经系统的正常发育和功能在很大程度上依赖于常驻巨噬细胞（称为小胶质细胞）的活性。小胶质细胞具有异质性和多功能性，可在脊髓和大脑内实现不同的基因表达和行为。许多研究探索了脑小胶质细胞的功能，并广泛地详细介绍了纯化方法。然而，小鼠脊髓中小胶质细胞的纯化缺乏全面的描述。相比之下，与未精制的提取物相比，使用高度纯化的胶原酶在中枢神经系统组织中缺乏报告。在这项研究中，从 8-10 周龄的 C57BL/6 小鼠身上切除脊柱和脊髓。随后的消化采用高度纯化的胶原酶，小胶质细胞纯化采用密度梯度。对细胞进行流式细胞术染色，通过 CD11b 和 CD45 染色评估活力和纯度。结果显示，平均存活率为80%，平均纯度为95%。总之，小鼠小胶质细胞的操作涉及用高度纯化的胶原酶消化，然后是密度梯度。这种方法有效地产生了大量的脊髓小胶质细胞群。

The defining characteristic of vertebrates is the vertebral column or spine, in which the notochord has been replaced by a sequence of segmented bones called vertebrae, divided by intervertebral discs. This succession of osseous material shapes the spinal canal, a cavity that encloses and protects the spinal cord1. In the genus Rodentia, the spine is usually formed by seven cervical vertebrae, thirteen thoracic vertebrae, six lumbar vertebrae, and a variable number of caudal vertebrae2,3. The length of the spinal cord is similar to that of the spine, and the terminal filum is a non-nervous structure that anchors the spinal cord to the sacrum. Additionally, nerve fibers exit through the intervertebral foramen1.
The development and proper function of the central nervous system in mammals critically depend on the activity of the nervous system&#39;s resident macrophages, called microglia4. Although microglia were initially described as brain resident phagocytes, recent research has attributed many dynamic functions to these cells5,6. Microglia&#39;s size ranges from 7 to 10 &#181;m in homeostasis; they are considered among the most versatile cells in the body and can adapt morphologically and functionally to their constantly changing environment7. These cells exhibit high heterogeneity during both the embryonic and adult stages8,9, while in the adult stage, they also display complex functional heterogeneity based on their spatiotemporal context10. The heterogeneity and multiple functions of microglia allow for differential gene expression and behavior in the spinal cord and brain. It has been shown that CD11b, CD45, CD86, and CCR9 expression is higher in the spinal cord compared to the brain8,9.
Multiple protocols exist for cerebral microglia isolation11,12; however, only a few exist for spinal cord microglia13,14. Optimizing a method for purifying microglia from the spinal cord facilitates the development of multiple studies focused on discovering microglia physiology. This protocol aims to describe a simple and highly reproducible extraction of the mouse spinal cord and the purification of microglia (Figure 1).

作者聚焦：小胶质细胞对脊髓异质性和纯化的研究 

小鼠脊髓小胶质细胞的快速高效富集

Our research focuses on the heterogeneity of microglia, which can exhibit both pro and anti-inflammatory behaviors across different pathologies and regions like the brain and spinal cord. We aim to enable a deeper understanding of spinal cord microglia behavior in diverse scenarios. The heterogeneity of microglia has made it difficult to classify them.This year, a new and broad classification has been proposed that goes beyond just a pro or anti-inflammatory status. Currently, achieving high purity and gel in isolating spinal cord microglia from animal models of neurological pathologies is a significant experiment and challenge. We identified a novel cytokine signaling pathway in the spinal cord microglia during chronic pain.Our protocol allows for a more controlled and somatic digestion, followed by a density gradient. Both result in less contamination by astrocytes compared to currently employed techniques for the purification of microglia from the spinal cord, brain, or embryos.

利用小鼠脊髓组织，使用高度富含胶原酶和热溶菌素的混合物进行酶消化。所得的消化组织通过40μm过滤器以去除未消化的物质。收集的细胞通过Percoll密度梯度富集，下部为90%，上部为45%。然后用CD45和CD11b抗体对界面内富含小胶质细胞的细胞进行染色，并进行流式细胞术分析（图1）。
从枕骨区域延伸到骶骨的脊柱解剖涉及椎旁肌肉组织的暴露和解剖。?...

Watch this Scientific Journal Video about Microglia Research on Spinal Cord Heterogeneity and Purification at JoVE.com

小胶质细胞被认为是体内最通用的细胞之一，能够进行形态和功能适应。它们的异质性和多功能性能够维持大脑稳态，同时也与各种神经系统病理有关。在这里，描述了一种净化脊髓小胶质细胞的技术。

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

我们的研究重点是小胶质细胞的异质性，它可以在不同的病理和区域（如大脑和脊髓）中表现出促炎和抗炎行为。我们的目标是更深入地了解脊髓小胶质细胞在不同情况下的行为。小胶质细胞的异质性使得很难对它们进行分类。今年，提出了一种新的、广泛的分类，它超越了促炎或抗炎状态。目前，从神经病理学动物模型中分离脊髓小胶质细胞时实现高纯度和凝胶化是一项重大的实验和挑战。我们在慢性疼痛期间在脊髓小胶质细胞中发现了一种新的细胞因子信号通路。我们的方案允许更可控的体细胞消化，然后是密度梯度。与目前用于从脊髓、大脑或胚胎中纯化小胶质细胞的技术相比，两者都减少了星形胶质细胞的污染。

rapid-and-efficient-enrichment-of-mouse-spinal-cord-microglia

Research

JoVE Journal

Neuroscience

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

1.2K 次观看.  Mexico Children's Hospital. 我们的研究重点是小胶质细胞的异质性，它可以在不同的病理和区域（如大脑和脊髓）中表现出促炎和抗炎行为。我们的目标是更深入地了解脊髓小胶质细胞在不同情况下的行为。小胶质细胞的异质性使得很难对它们进行分类。今年，提出了一种新的、广泛的分类，它超越了促炎或抗炎状态。目前，从神经病理学动物模型中分离脊髓小胶质细胞时实现高纯度和凝胶化是?...