Name: isolation and characterization of the immune cells from micro-dissected mouse choroid plexuses
Uploaded: 2022-02-03T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses at JoVE.com

我们感谢巴斯德动物研究所和CB-UTechS设施成员的帮助。这项工作得到了巴斯德研究所的财政支持。

所有程序均符合欧盟委员会关于处理实验动物的指南，指令86/609/EEC。它们由第59号伦理委员会批准，由CETEA / CEEA第089号批准，编号为dap210067和APAFIS #32382-2021070917055505 v1。
1. 材料的准备
<ol><li>将所有抗体（材料表）储存在4°C，避免光照。</li>
	<li>DAPI储备溶液（1mg / mL）：将粉末重悬于PBS-/- （材料表），等分试样，并储存在-20°C。...

<ol>
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旨在了解对大脑稳态和疾病的免疫学贡献的研究主要集中在脑实质内的细胞上，而忽略了脑边界，如CP，但这些边界仍然是大脑功能的关键贡献者2，3。由于CP的小尺寸，驻留免疫细胞的数量少以及访问这种组织的复杂，CP免疫细胞群的分析具有挑战性。对全脑免疫细胞（CD45 +）进行的流式细胞术不允许表征驻留在CP中的罕见免疫群体。为了高精?...

自从保罗·埃尔利希（Paul Erhlich）在19世纪末 发现血脑屏障以来，大脑一直被认为与其他器官和血液几乎分离。然而，在过去的十年中，出现了大脑功能由各种生物因素塑造的概念，例如肠道微生物群和全身免疫细胞和信号1，2，3，4。与此同时，其他脑边界，如脑膜和脉络丛（CP）已被确定为活动免疫 - 脑串扰的界面，而不是惰性屏障组织5，6，7，8。
脑瘫构成血脑脊液屏障，是分隔大脑和外围的边界之一。它们位于大脑的四个心室中，即第三个、第四个和两个侧心室，并且与参与神经发生的区域（例如海马体的脑室下区和粒下区）相邻3。在结构上，CP由由单层上皮细胞包围的开窗状毛细血管网络组成，这些细胞通过紧密和粘附连接相互连接9，10。脑瘫上皮的主要生理作用涉及脑脊液的产生，脑脊液从废物代谢物和蛋白质聚集体中冲洗大脑，以及产生和控制各种信号分子的血脑通道，包括激素和神经营养因子11，12，13。来自CP的分泌分子通过调节神经发生和小胶质细胞功能来塑造大脑的活动14，15，16，17，18，19，这使得CP对大脑稳态至关重要。CP还从事各种免疫活动;虽然在非病理条件下脑实质中的主要免疫细胞类型是小胶质细胞，但CP免疫细胞群的多样性与外周器官一样广泛3，7，这表明免疫调节和信号传导的各种通道在CP中起作用。
内皮细胞和上皮细胞之间的空间，CP基质，主要由边界相关巨噬细胞（BAM）填充，其表达促炎细胞因子和与抗原呈递相关的分子，以响应炎症信号3。巨噬细胞的另一种亚型，Kolmer's epiplexus细胞，存在于CP上皮的顶端表面20。CP基质也是树突状细胞，B细胞，肥大细胞，嗜碱性粒细胞，嗜中性粒细胞，先天淋巴细胞和T细胞的利基，这些细胞主要是能够识别中枢神经系统抗原的效应记忆T细胞7，21，22，23，24。此外，CP处免疫细胞群的组成和活性会随着全身或脑部的扰动而变化，例如在衰老过程中10，14，15，21，25，微生物群扰动7，stress26和疾病27，28。值得注意的是，这些变化被认为间接塑造了大脑功能，即CP CD4 + T细胞向Th2炎症的转变发生在大脑衰老中，并触发CP的免疫信号传导，可能塑造衰老相关的认知能力下降14，15，21，25，29.因此，阐明CP免疫细胞的特性对于更好地了解它们对CP上皮生理和分泌的调节功能至关重要，从而破译它们在健康和疾病条件下对大脑功能的间接影响。
脑瘫是仅包含少数免疫细胞的小结构。它们的隔离需要在灌注的初步步骤后进行显微解剖;否则，血液中的免疫细胞将构成主要污染物。该协议旨在使用流式细胞术表征CP的髓细胞和T细胞亚群。该方法鉴定了在非炎性条件下构成小鼠CP的约90%的免疫细胞群，根据最近发表的使用其他方法解剖免疫CP异质性的工作7，10，28。该方案可用于表征CP免疫细胞区室中疾病的变化以及 体内其他实验范式。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Albumin, bovine</td>
			<td>MP Biomedicals</td>
			<td>160069</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>APC anti-mouse CX3CR1</td>
			<td>BioLegend</td>
			<td>149008</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse TCRb</td>
			<td>BioLegend</td>
			<td>109212</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse IA-IE</td>
			<td>BioLegend</td>
			<td>107628</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5 Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry analyzer</td>
		</tr>
		<tr>
			<td>BV711 anti-mouse Ly6C</td>
			<td>BioLegend</td>
			<td>128037</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>Enzyme to dissociate CP tissue</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Live/dead marker</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td>Ion chelator</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>FST</td>
			<td>14058-11</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45</td>
			<td>BioLegend</td>
			<td>103108</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Flow controller infusion inset</td>
			<td>CareFusion</td>
			<td>RG-3-C</td>
			<td>Blood perfusion inset</td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11018-12</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>Ketamine, anesthesic</td>
		</tr>
		<tr>
			<td>OneComp eBeads</td>
			<td>Invitrogen</td>
			<td>01-1111-42</td>
			<td>Control beads to realize compensation</td>
		</tr>
		<tr>
			<td>PBS-/-</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PBS+/+</td>
			<td>Gibco</td>
			<td>14040-091</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100708</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80</td>
			<td>BioLegend</td>
			<td>123110</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE-Dazzle 594 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101256</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, anesthesic</td>
		</tr>
		<tr>
			<td>thin forceps</td>
			<td>Dumoxel Biology</td>
			<td>11242-40</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Vetergesic</td>
			<td>Ceva</td>
			<td></td>
			<td>Buprenorphin, analgesic</td>
		</tr>
	</tbody>
</table>

isolation and characterization of the immune cells from micro-dissected mouse choroid plexuses

大脑不再被视为孤立运作的器官;越来越多的证据表明，外周免疫系统的变化可以间接塑造大脑功能。在大脑和体循环之间的界面，构成血脑脊液屏障的脉络丛（CP）被强调为脑外交流的关键部位。脑脊髓质酸产生脑脊液、神经营养因子和信号分子，这些分子可以塑造大脑稳态。CP也是一个活跃的免疫学利基。与在生理条件下主要由小胶质细胞填充的脑实质相反，CP免疫细胞的异质性概括了在其他外周器官中发现的多样性。脑瘫免疫细胞的多样性和活性随衰老、应激和疾病而变化，调节脑瘫上皮的活性，从而间接塑造大脑功能。该协议的目标是分离小鼠CP并确定填充它们的主要免疫亚群的约90%。该方法是表征CP免疫细胞并了解它们在协调外围到大脑通信中的功能的工具。拟议的方案可能有助于破译CP免疫细胞如何在健康和各种疾病条件下间接调节大脑功能。

这里介绍的流式细胞术分析以高度可重复的方式成功地揭示了髓细胞和T细胞的主要亚群（分别为图1和图2），以及它们每只小鼠的相对总数（图3）。
对骨髓细胞的流式细胞术分析表明，CP由CD11b + CX3CR1 + F4 / 80high BAM填充，占CP处CD45 +免疫细胞的近80%。这些BAM根据其MHC-II...

Watch this Scientific Journal Video about Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses at JoVE.com

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

本研究使用流式细胞术和两种不同的门控策略对分离的灌注小鼠脑脉络丛;该协议确定了填充该大脑结构的主要免疫细胞亚群。

从微解剖小鼠脉络丛中分离和表征免疫细胞

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

该协议可能有助于解决有关脉络丛和外周免疫细胞在小鼠模型中各种情况下调节大脑中的作用的问题。该协议允许对小鼠脉络膜丛中独特的免疫细胞群进行深入的定量和定性分析。由于脉络膜丛的体积小且颜色透明，在开始时可能很难解剖脉络丛。我们建议先在非灌注动物身上练习。首先，将C57黑色六只小鼠背侧解剖的大脑放在培养皿中，并将培养皿放在双眼环的目标下方。在使用镊子将大脑保持在适当的位置时，将另一对镊子的两端向下插入半球之间的中线。使用镊子，将带有胼胝体和海马体的皮层从隔膜上拉开，暴露外侧心室和第三脑室的一部分。将外侧脉络丛识别为外侧心室内衬的长面纱，两端有喇叭形。然后使用薄镊子的两端，收集侧脉络丛。小心收集被海马体后褶皱隐藏的三角形后部。接下来，将带有对侧半球的胼胝体和海马体的皮层从隔膜上拉开，以暴露整个第三脑室和对侧心室。使用细镊子，收集第三脉络丛，其标识为具有颗粒状表面的短结构。然后收集另一个侧脉络丛。接下来，将镊子的两端插入小脑和中脑之间，并将小脑与脑桥和髓质分离以暴露第四脑室。然后将第四脉络丛确定为具有颗粒状表面的长球状结构，该表面面从小脑和髓质之间的外侧右端到左端排列第四脑室。使用细镊子，收集第四脉络丛。用胶原酶IV孵育所有收集的脉络膜丛后，轻轻地上下移液约10次以完成解离。然后用MACS缓冲液将试管填充至1.5毫升以停止胶原酶IV活性。接下来，离心细胞并丢弃上清液，然后用1.5毫升MACS缓冲液洗涤细胞。加入适当的补偿微珠和抗体溶液后，将样品在冰上孵育30至45分钟，保护它们免受光照。然后用1.5毫升MACS缓冲液填充试管，并离心细胞和补偿微球。将细胞和补偿珠重悬于500微升MACS缓冲液中后，通过70微米过滤器过滤细胞。打开流式细胞仪和软件后，根据大小选择前向散射区域与侧面散射区域和细胞门。然后为具有前向散射面积与前向散射高度的单个单元创建子门。接下来，为具有DAPI与前向散射区域的活细胞创建子门，以排除DAPI阳性细胞。然后为CD45阳性免疫细胞创建一个子门，CD45与前向散射区域。现在，从CD45阳性细胞创建一个子门，并选择F480与CD11b来识别CD11b阳性F480阳性和CD11b阳性F480阴性群体。然后为CD11b阳性F480阳性细胞创建子门，并选择CX3CR1与IA-IE，以鉴定CX3CR1阳性IA-IE阳性边界相关巨噬细胞和CX3CR1阳性IA-IE阴性巨噬细胞。接下来，为 CD11b 阳性 F480 阳性 CX3CR1 阳性 IA-IE 阴性巨噬细胞创建子门，并选择 F480 与 CD11b 以鉴定 CD11b 高 F480 中间 CX3CR1 阳性 IA-IE 阴性 Kolmer 氏双神经丛巨噬细胞和 CD11b 阳性 F480 阳性 CX3CR1 阳性 IA-IE 阴性边界相关巨噬细胞。然后从CD11b阳性F480阴性人群中，门为Ly6C与IA-IE。选择主要对应于单核细胞或嗜中性粒细胞的IA-IE阳性Ly6C阴性群体和IA-IE阴性Ly6C阳性细胞。最后，为 CD11b 阳性 F480 阴性 IA-IE 阳性 Ly6C 阴性群体创建子门，并选择 CD11b 与 CX3CR1。然后鉴定CD11b高CX3CR1低和CD11b阳性CX3CR1阴性人群。对于 T 细胞门控，如前所述排除 DAPI 阳性细胞后，使用 CD45 与 FSC-A 为 CD45 阳性免疫细胞创建子门，然后从 CD45 阳性细胞创建子门，选择 TCR β 与 CD11b。最后，为TCRβ阳性CD11b阴性群体创建子门，并选择CD4与CD8a。鉴定 CD8 阴性和 CD4 阳性 T 细胞以及 CD8 阳性 CD4 阴性 T 细胞。这里展示的流式细胞术分析以高度可重复的方式成功地揭示了骨髓细胞和T细胞的主要亚群及其每只小鼠的相对总数。对髓系细胞的流式细胞术分析表明，脉络丛由CD11b阳性CX3CR1阳性F480高边界相关巨噬细胞填充，占脉络丛CD45阳性免疫细胞的80%。还鉴定了科尔默氏双神经丛巨噬细胞的CD11b高F480中间体CX3CR1阳性IA-IE阴性群体。在CD11b阳性F480阴性免疫细胞中，表征了两个不同的Ly6C阴性IA-IE阳性细胞群体，可能对应于树突状细胞。T细胞的分析和门控策略突出了CD45阳性CD11b阴性TCRβ阳性细胞的次要群体的存在。其中，每只小鼠约30-50个CD8阴性CD4阳性和CD8阳性CD4阴性T细胞在生理条件下填充脉络丛。控制灌注质量很重要，因为任何血液污染都可能模糊脉络丛的免疫成分。该方法之后可以对选定的细胞群进行分选，以进行进一步分析，例如批量或单细胞RNA测序。

Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

科研

生物学

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Characterization of Microvesicles from Peripheral Blood

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

成年小鼠黏膜下层和固有层肠道胶质细胞的分离

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...