Name: flow cytometric analysis of lymphocyte infiltration in central nervous system during experimental autoimmune encephalomyelitis
Uploaded: 2020-11-17T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis at JoVE.com

这项研究得到了国家自然科学基金资助（31570921至ZJ，81571533至LS），上海市卫生委和计划生育委员会（201540206至ZJ），瑞金医院北研究资助（2017ZX01至ZJ）。

这里描述的所有方法都经过了上海交通大学基础医学学院动物委员会的批准。
1. 材料的准备
<ol><li>使用MOG35-55的 MEVGWYRSPFSRVVHLYRNGK 序列从商业来源获得冻干肽。确保肽的纯度为 >95%。在磷酸盐缓冲盐水 （PBS） 中准备 10 mg/mL MOG 库存溶液，并储存在 -20°C。</li>
	<li>将一个100毫克的M.结核病H37 Ra放入25 mL的完全弗伦德佐剂M. tuberculosis（CFA）和混合中，准备4mg/mL的M.结?...

<ol>
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本研究提出了在C57BL/6小鼠中使用MOG35-55诱导和监测EE的方案，该模型被认为是MS的典型神经免疫实验动物模型。 EAE可以根据研究的目的，诱导改变小鼠的菌株或用于诱导的蛋白质类型。例如，在SJL小鼠中使用PLP139~151肽可以诱发复发-复发的EE疾病过程，这特别适合评估复发15的治疗效果。此处概述的实验程序也可以应用于其他 EAE 协议7。在此模型中，C57BL/6小鼠接?...

作为一种慢性脱髓性疾病，MS影响全球约250万人，缺乏治疗1。它也被认为是一种自身免疫性疾病，其中明蛋白抗原特异性T淋巴细胞引发炎症反应，导致在CNS2中脱叶和结子损伤。实验自身免疫性脑脊髓炎（EAE）作为CNS 3中典型的自身免疫性脱毛病模型，已被广泛用于研究MS的致病机制。有两种方法可以诱导EE：一种是积极诱导EE，通过免疫动物的脑素成分，另一种是采用转移，通过转移脑原T细胞到受体2，4，5。,4,5对EAE的感知性在不同的动物菌株6中是不同的。在C57BL/6小鼠中，霉素奥利戈登德细胞蛋白（MOG）35~55挑战诱导一种单相疾病，在CNS中具有广泛的脱霉和炎症，该病在基因靶向小鼠7的实验中经常使用。
生成脑电图特异性活性T细胞是EE疾病的发生和发展所需的，是EE和MS的免疫学标志。当MOG 35-55 Ag遇到时，这些T淋巴细胞诱导炎症和诱导效应细胞进入中枢，导致去叶化和轴翁破坏88，99。在EAE模型中，有充分证据表明神经抗原特异性CD4+T细胞可以启动和维持神经炎和病理学3,3，10。根据主要细胞因子的产生，CD4+ T淋巴细胞被分为不同的子集：Th1（以产生干扰素-γ）、Th2（以白细胞间4的产生为特征）和Th17（以白细胞间17的产生为特征）。据认为，Th1和Th17细胞的激活有助于诱导，维持和调节炎症消炎在EAE和MS通过分泌效应细胞因子 IFN-γ和IL-17，这能够激活巨噬细胞和招募嗜中性粒细胞到炎症部位加速病变11。
由于自反应T细胞将BBB穿过到中枢，诱导MS和EE的疾病发展，因此分析CMS中的T细胞非常重要。然而，很少有既定的协议，从中枢12分离淋巴细胞。因此，开发了一种优化的方法，用于从大脑中隔离单核细胞，用标记CD45、CD11b、CD3、CD4、INF-g和IL-17分析T淋巴细胞，用于流细胞学。该方法使用MOG35~55辅助结核分 枝杆 菌H37 Ra和百日咳毒素工作溶液（PTX）在小鼠中诱导EE的主动免疫模型。然后，采用机械分离和密度梯度离心方法分离CNS单核细胞。最后，通过染色多个标记来识别大脑中的T淋巴细胞和子集，优化了流式细胞测量门控策略。

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor700 anti-mouse CD45.2</td>
			<td>eBioscience</td>
			<td>56-0454-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/CD32 Fc block</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-g</td>
			<td>eBioscience</td>
			<td>17-7311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa X-20</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>Wheaton</td>
			<td>353107542</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) (500X)</td>
			<td>eBioscience</td>
			<td>00-4975-03</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Intracellular Fixation &#38; Permeabilization Buffer Set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD3</td>
			<td>BioLegend</td>
			<td>100203</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400605</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#39;s Adjuvant Complete (CFA)</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG2a kappa Isotype Control (eBM2a), Alexa Fluor 700, eBioscience</td>
			<td>eBioscience</td>
			<td>56-4724-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37 Ra</td>
			<td>Difco Laboratories</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse IL-17A</td>
			<td>eBioscience</td>
			<td>12-7177-81</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100422</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400617</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400631</td>
			<td></td>
		</tr>
		<tr>
			<td>pertussis toxin (PTX)</td>
			<td>Sigma-Aldrich</td>
			<td>P-2980</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control (eBRG1), APC, eBioscience</td>
			<td>eBioscience</td>
			<td>17-4301-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control (eBR2a), PE, eBioscience</td>
			<td>eBioscience</td>
			<td>12-4321-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat MOG35&#8211;55 peptides</td>
			<td>Biosynth International</td>
			<td></td>
			<td>MEVGWYRSPFSRVVHLYRNGK</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of lymphocyte infiltration in central nervous system during experimental autoimmune encephalomyelitis

多发性硬化症（MS）是由环境因素和易感遗传背景共同作用的中枢神经系统（CNS）的自身免疫性疾病。实验性自身免疫性脑脊髓炎（EAE）是MS的典型疾病模型，广泛用于研究T淋巴细胞特异性骨髓抗原在中枢素引起炎症反应的发病机制。评估中枢子中的淋巴细胞如何调节疾病的发展是非常重要的。然而，由于难以从大脑中隔离和检测渗透淋巴细胞，测量中枢机中渗透淋巴细胞的数量和质量的方法非常有限。本手稿提供了一个协议，用于从 CNS 中分离、识别和表征渗透淋巴细胞，有助于我们了解淋巴细胞如何参与中枢自免疫性疾病的发展。

在对C57BL/6小鼠进行免疫接种后，所有小鼠每天进行称重、检查和评分，以发现神经体征。EAE 的代表性临床过程应导致图2A中所示的疾病曲线和图2B中所示小鼠体重的变化。C57BL/6用MOG35-55免疫的小鼠通常在第10-12天左右开始出现疾病症状，并在主动免疫后第15-21天左右达到疾病高峰（图2A）。重量变化是 EAE 模型中的一个有价值的指?...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis at JoVE.com

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

本手稿提供了在小鼠中诱导活性实验性自身免疫性脑脊髓炎 （EAE） 的协议。还提出了一种分离和表征中枢神经系统（CNS）中渗透淋巴细胞的方法，以表明淋巴细胞如何参与中枢自身免疫性疾病的发展。

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

我们在这里介绍的协议将有助于我们了解淋巴细胞如何参与中枢神经系统自身免疫性疾病的发展。用这种方法，可以按细胞研究大脑中的淋巴细胞。该协议也适用于分析免疫细胞特征和功能所需的其他模型和中枢神经系统疾病。在这段视频中，该协议可以很容易地演示如何诱导模型和分离大脑中的单细胞。在确认对踏板反射缺乏反应后，使用一毫升注射器将麻醉的 C57BL/6 小鼠注射 100 微克乳化 MOG 35-55，在 100 微升完全 Freund 的辅助剂中，将完全 Freund 的辅助剂注射到颈部附近的两个不同位置。在同一天和免疫后的第二天，内向小鼠注射200微升，每微升百日咳毒素工作溶液1纳米。第二次注射后，用加热垫将小鼠转移回其家笼并监测直至完全卧化。第二天和整个疾病过程中，检查所有小鼠，并盲目地检查和分级，检查表上概述的神经体征和体重的任何变化。在适当的实验终点点，小心地将颅骨从鼻子切到颈部，将每个动物的大脑从颅箱转移到含有10毫升RPMI介质的50毫升管中。将管子混合好，去除粘附的红血球，并通过吸气去除介质。在每个管子中加入10毫升的新鲜介质，并将大脑转移到单独的100毫米的盘子中。最后用剃须刀切开每个大脑，用塑料移液器将尽可能多的组织从一盘和六毫升的培养皿中转移到冰冷七毫升的玻璃中均质器中。使用软柱塞的松散柱塞研磨大脑，然后使用紧柱塞将组织带到，直到悬浮液是均匀的。然后，将产生的组织浆倒入冰上预冷却的 15 毫升锥形管中。当所有样品均质化后，用新鲜介质将每管的体积调整为7毫升，然后将每个组织悬浮液添加到一个新的冷冻15毫升管中，每管含有3毫升100%基底膜基质。通过反转混合几次，并使用三毫升移液器仔细缓慢地在每个组织溶液样品下添加一毫升 70% 密度梯度溶液。通过密度梯度离心分离细胞，并移除几乎所有的顶层，注意完全去除所有的米林。将接口转移到新的 15 毫升管中，用新鲜介质将音量调节到 10 毫升。然后再次离心样品，取出上先液，然后将颗粒重新插入每管约100微升的介质中。对于收获的脑细胞的流动细胞测量分析，将大约2倍10的6个细胞分配到96孔板的单个孔中。当所有细胞都镀光后，将100微升的500X细胞刺激鸡尾酒加上蛋白质运输抑制剂加入井中，并将板放在细胞培养培养箱中4小时。在孵化池结束时，通过离心和重新泵化每个孔100微升流动细胞仪缓冲液中的颗粒，将井底的细胞重新填充。用三个微升的抗小鼠CD16/CD32 Fc块预孵育细胞，在4摄氏度下预孵10分钟，然后染色。阻止任何非特定 Fc 调解的交互。在孵化结束时，将感兴趣的抗体鸡尾酒添加到每一个井中，并将板放在四摄氏度的温度下，防止光线。30分钟后，用每口井100微升流动细胞体缓冲液清洗井，通过离心沉淀细胞。丢弃超钠后，向每井添加200微升细胞内固定缓冲液。在室温下孵育30至60分钟后，通过离心收集样品，并在每井1倍渗透缓冲液的200微升中重新填充颗粒。再次将样品离心，在丢弃上流液后，用新鲜渗透缓冲液将颗粒重新在 100 微升中。接下来，加入适当的抗体，以检测任何感兴趣的细胞内抗原，在4摄氏度下孵育30分钟，不受光线影响。在孵化结束时，向每井添加100微升1X渗透缓冲液，通过离心将样品向下旋转。然后，在200微升流动细胞测量染色缓冲液中重新填充染色细胞，并根据标准协议通过流式细胞仪分析细胞。EAE 的典型临床过程应导致疾病曲线和体重变化，如图所示。C57BL/6小鼠接种MOG 35-55疫苗通常开始出现疾病症状约第10天至第12天，并在第15至21天左右达到疾病高峰。在发病前，免疫小鼠的体重逐渐增加，然后随着疾病症状的增加而减少。然而，小鼠通常不能完全恢复，通常继续发展单相慢性病病理学。正如这个具有代表性的流动分析所表明的，干扰素伽马产生Th1和IL17产生Th17细胞在EAE小鼠中显著增加。最重要的步骤是 3.10。操作员应转移中间层和光线，小心地尽可能少地使用其他层。按照这些协议，我们可以进一步T淋巴细胞的进一步转录分析研究

Research

JoVE Journal

Immunology and Infection

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

科研

免疫与感染

Culturing Microglia from the Neonatal and Adult Central Nervous System

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R Cross-linking

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...