Name: isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells
Uploaded: 2020-05-19T14:45:00
Description: Watch this Scientific Journal Video about Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells at JoVE.com

作者感谢达特茅斯比较医学和研究中心（CCMR）的工作人员对用于这些研究的小鼠的专业护理。伯恩斯坦研究基金资助了这项研究。

所有动物工作都采用达特茅斯盖泽尔医学院机构动物护理和使用委员会（IACUC）审查和批准的协议。
1. 处理脑和脊髓样本进行脱钙
<ol><li>分离脑和脊髓样本
	<ol><li>通过吸入CO2 对小鼠实施安乐死。确保 CO2 流速每分钟置换保持架容积的 10%–30%。</li>
		<li>使用镊子提起剑突，并用剪刀在肋骨下方横向切割腹壁，向上拉以避免切割下面的血管或器官?...

在稳态和疾病期间评估CNS区室细胞组成的方法对于了解CNS的生理和病理状态至关重要。然而，尽管是中枢神经系统的重要屏障并容纳多种免疫细胞，但脑膜经常被省略在分析中，因为许多传统的大脑和脊髓组织提取方法不允许收集这些膜。这种遗漏是我们了解脑膜细胞组成和功能及其在稳态和炎症条件下的作用的关键限制。最近的研究表明，驻留在脑膜隔室中的常驻和外周来源的免疫细胞在维持?...

中枢神经系统（CNS）是一个免疫学专业化的部位。中枢神经系统实质（不包括脑脊液间隙、脑膜和脉管系统）通常被视为免疫特权部位 1,2,3,4,5，在稳态条件下相对缺乏免疫细胞 2,6,7。相比之下，由硬脑膜、蛛网膜和软脑膜层组成的脑膜是中枢神经系统隔室的重要组成部分，在疾病发病机制期间积极参与稳态免疫监视和炎症过程 3,6,7,8。在稳态条件下，脑膜支持许多免疫哨兵细胞，包括先天淋巴细胞（ILC），巨噬细胞，树突状细胞（DC），肥大细胞，T细胞，以及在较小程度上的B细胞9,10,11。
脑膜是高度血管化的结构，含有淋巴管，在中枢神经系统与其外围之间提供淋巴连接8,12,13,14。在由中枢神经系统损伤、感染、自身免疫甚至神经变性引起的炎症性疾病中，外周来源的免疫细胞会浸润脑实质并改变脑膜内的免疫景观。细胞浸润后，脑膜可能代表外周来源免疫细胞的功能生态位，促进免疫细胞聚集、局部免疫细胞活化和中枢神经系统区室的长期存活。在影响中枢神经系统的多种疾病中观察到明显的脑膜炎症，包括多发性硬化症 （MS）15,16,17,18,19、中风 20,21、无菌损伤 22,23（即脊髓损伤和创伤性脑损伤）、偏头痛 24 和微生物感染 25,26，27,28,29.因此，脑膜区室中常驻细胞和外周衍生免疫细胞的表征对于了解这些细胞在稳态条件和疾病发病机制中的作用至关重要。
从颅骨和椎体中提取大脑、脊髓和脑膜在技术上具有挑战性且耗时。目前还没有可用于快速提取大脑的技术，所有三个脑膜层都完好无损。虽然椎板切除术产生出色的脊髓组织形态并保留了脑膜层，但它既非常耗时又复杂30,31。相反，更传统的提取方法，例如从颅骨中取出大脑和脊髓的液压挤出有助于快速提取CNS组织，但蛛网膜和硬脑膜都因这些技术而丢失30,31。在脑和脊髓组织的常规分离过程中，硬脑膜和蛛网膜层的遗漏导致对中枢神经系统隔室内细胞的分析不完整。因此，确定专注于快速提取具有完整脑膜的CNS组织的新技术对于CNS室的最佳分析至关重要。
本手稿介绍了两种从小鼠中快速提取大脑、脊髓和脑膜的方法，有助于对中枢神经系统实质和脑膜中的常驻细胞和外周衍生免疫细胞进行下游分析。这些优化的方案侧重于 1） 分离单细胞悬液以进行下游分析和 2） 准备组织以进行组织学处理。从大脑、脊髓组织以及硬脑膜和蛛网膜脑膜32 获得单细胞悬浮液可以同时分析存在于实质和脑膜隔室中的细胞。单细胞悬浮液可用于不同的应用，包括用于体外刺激的细胞培养测定33、酶联免疫斑点（ELISpot）28、34、35、流式细胞术36、33和单细胞37或批量转录组学。此外，分别具有完整颅骨或椎体的整个大脑和脊髓脱钙的优化方案允许周围骨骼的温和脱钙，使脑膜完整并保留组织形态。该方法允许在实质和脑膜间隙内使用免疫组织化学（IHC）或原位杂交（ISH）技术选择性鉴定蛋白质或RNA。中枢神经系统内驻留细胞和外周来源免疫细胞的表型、活化状态和定位的表征可能为了解中枢神经系统区室中的单个细胞类型如何促进稳态和疾病发病机制提供必不可少的信息。

<table>
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>37002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-12R centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 15 mL</td>
			<td>VWR</td>
			<td>525-1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Hauser Scientific</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>VWR</td>
			<td>18000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11003-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene tube, 14 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Fisher Scientific</td>
			<td>NC0595256</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Durmont #7Forceps</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Amresco</td>
			<td>0105-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter top tube, 5 mL</td>
			<td>VWR</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable viability stain 780</td>
			<td>Becton Dickinson</td>
			<td>565388</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>Gallios</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (488 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-546-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (594 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-586-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 488</td>
			<td>Jackson immunoresearch</td>
			<td>111-545-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 594</td>
			<td>Jackson immunoresearch</td>
			<td>112-585-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 650</td>
			<td>Jackson immunoresearch</td>
			<td>112-605-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balnced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Andwin Scientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13004-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher chemical</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4 (anhydrous)</td>
			<td>Sigma Aldrich</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse FC block (CD16/32)</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HP04 (anhydrous)</td>
			<td>Fisher Chemical</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher chemical</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 25 gauge</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal mouse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>31881</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh strainer</td>
			<td>VWR</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td>Diluted to 4% using 1 x PBS</td>
		</tr>
		<tr>
			<td>Pasteur pipette, 9 inch, unplugged</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Rat Anti-Mouse CD4</td>
			<td>Becton Dickinson</td>
			<td>553730</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 Rat Anti-Mouse CD19</td>
			<td>Becton Dickinson</td>
			<td>562329</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll density gradient media</td>
			<td>GE healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 Rat Anti-Mouse CD45</td>
			<td>Becton Dickinson</td>
			<td>550994</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 100 mm</td>
			<td>VWR</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher Scientific</td>
			<td>13-636-AB150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Corporation</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 200 &#181;l</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 1 mL</td>
			<td>USA Scientific</td>
			<td>1111-2831</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#181;l</td>
			<td>USA Scientific</td>
			<td>1111-1816</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, 1 mL</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Diamond mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse CD3 (SP7 clone)</td>
			<td>Abcam</td>
			<td>ab16669</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse laminin</td>
			<td>Abcam</td>
			<td>ab11575</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse ERT-R7</td>
			<td>Abcam</td>
			<td>ab51824</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 1 mL</td>
			<td>VWR</td>
			<td>357521</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 10 mL</td>
			<td>VWR</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 5 mL</td>
			<td>VWR</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors, extra fine</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Becton Dickinson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter system</td>
			<td>Millipore</td>
			<td>20207749</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum flask</td>
			<td>Thomas Scientific</td>
			<td>5340-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum in-line filter</td>
			<td>Pall Corporation</td>
			<td>4402</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum line</td>
			<td>Cole Palmer</td>
			<td>EW-06414-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ThermoFisher Scientific</td>
			<td>Versa bath</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells

中枢神经系统（CNS）由大脑和脊髓组成，被脑膜包裹，膜层是外围和CNS之间的屏障。中枢神经系统是一个免疫学特化的部位，在稳态条件下，免疫特权在中枢神经系统实质中最为明显。相比之下，脑膜含有多种常驻细胞，包括先天性和适应性免疫细胞。在由中枢神经系统损伤、自身免疫、感染甚至神经变性引发的炎症性疾病中，外周来源的免疫细胞可能会进入脑实质并居住在脑膜内。这些细胞被认为在中枢神经系统疾病发病过程中执行有益和有害作用。尽管有这些知识，但在分析中枢神经系统隔室时，脑膜经常被忽视，因为传统的中枢神经系统组织提取方法省略了脑膜层。该协议提出了两种不同的方法，用于快速分离鼠中枢神经系统组织（即脑，脊髓和脑膜），适用于通过单细胞技术，免疫组织化学和原位杂交方法进行下游分析。所描述的方法提供了对CNS组织的全面分析，非常适合评估在稳态条件下和疾病发病期间占据CNS区室的细胞的表型，功能和定位。

该代表性实验旨在量化B和T细胞，并描述B和T细胞在稳态条件下脑膜和实质CNS区室以及小鼠进行性MS模型（即TMEV-IDD）中的定位。如前所述，TMEV-IDD通过5 x 106 斑块形成单位（PFU）的TMEV BeAn的颅内感染在5周龄雌性SJL小鼠中诱导29。
本研究评估了感染后第120天慢性TMEV-IDD期间脑膜，大脑和脊髓中的B和T细胞。使用年龄匹配的假处理小鼠作为对照。该研究由?...

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Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

本文提出了两种优化的方案，用于检查中枢神经系统内的常驻和外周来源的免疫细胞，包括大脑、脊髓和脑膜。这些协议中的每一个都有助于确定在稳态和炎症条件下占据这些隔室的细胞的功能和组成。

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Rapid isolation of CNS tissues including the meninges allows for a comprehensive analysis of immune cell phenotype, function, and localization in homeostatic and pathogenic conditions. The two demonstrated methods allow for the quick extraction of CNS tissues with the meninges, ideal for downstream analysis using single cell techniques and histological methods. Although the representative results focus on the analysis of immune cells, these two methods can be used to analyze CNS resident cells, including microglia, astrocytes, pericytes, endothelial cells, stromal cells, and neurons.Begin by isolating brain and spinal cord samples from euthanized mice. After removing the head, make a midline incision in the skin and flip it over the eyes to free the skull. Cut at the nasal bone to release the mandible from the skull.Then remove the mandible, tongue, and eyes. Cut along the lateral aspects of the skull to release the tissue along the external auditory meatus and trim it. To collect the spinal cord sample, separate the rib cage from the spinal column by cutting parallel to the spine with sharp scissors.Make a small cut at the lower lumbar region to isolate the spinal column, then trim and remove any remaining muscle along the spine to expose the vertebrae. When performing decalcification, check the bone daily to determine if it is soft and pliable. Remove the tissue from the EDTA solution with forceps, place it on a Petri dish, and gently test the bone softness with a 25 gauge needle.If the needle easily penetrates the bone, the decalcification process is complete. To extract the skull cap in the brain, make a midline incision in the skin and flip the skin over the eyes. Place the scissors within the foramen magna and begin cutting the skull laterally along the cortices towards the olfactory bulb, keeping the incisions above the external auditory meatus and mandible.Perform the same cuts on the opposite side with cuts meeting at the olfactory bulb to free the skull cap from the brain. Using forceps, peel back the skull cap and place it into a 15 milliliter conical tube containing five milliliters of cold RPMI medium supplemented with 25 millimolar HEPES. Place curved forceps below the base of the brain and lift them to free the brain from the skull cap.Extract the vertebrae column and spinal cord tissue by cutting parallel to the spine to separate the rib cage from the spinal column. Then make a small cut at the lower lumbar region to isolate the vertebrae column. Trim and remove any remaining muscle along the spine to expose the vertebrae.Next, place the extra fine surgical scissors within the vertebral column and cut along the lateral edge of the column. Cut the opposite lateral edge completely to divide the vertebral column into an anterior and posterior portion. Use forceps to peel away the spinal cord slowly and carefully from the vertebral column and place it in a 15 milliliter conical tube containing five milliliters of cold RPMI with 25 millimolar HEPES.Then transfer the anterior and posterior portions of the spinal column to another tube. To remove the meninges from the skull cap, take the skull cap out of the RPMI media and use sharp forceps to score around the outer edge. Peel the meninges away from the edge of the skull cap, scraping to remove the dural and arachnoid meninges and place the meninges on a Petri dish.To remove the meninges from the vertebral column, take the column out of the media and score around the edges of the vertebral column with sharp forceps to free the meninges. Use curved forceps to peel away the meninges from the edge of the vertebrae and place the meninges on a Petri dish. Place a nylon mesh strainer in a 50 milliliter conical tube.To create a cell suspension, move the meninges into a strainer and add three milliliters of HEPES supplemented RPMI, then use a plunger from a five milliliter syringe to grind the tissue and media through the strainer. Pour the brain or spinal cord tissue with the media into a 100 millimeter Petri dish and use forceps to move the tissue to the bottom. Mince the brain tissue with a sterile razor blade and gather the tissue at the bottom of the plate.Add three milliliters of RPMI supplemented with 10%FCS to the Petri dish, then use a 10 milliliter serological pipette to resuspend the tissue in the media and transfer it to a 15 milliliter conical tube. Add collagenase type 1 and DNase 1 to the tissue and incubate the tubes in a 37 degree Celsius water bath for 40 minutes. Invert the tubes every 15 minutes to thoroughly mix the tissue with the enzymes.After incubation, add EDTA to each tube for a final concentration of 0.01 molar and incubate the tubes for an additional five minutes to inactivate the collagenase. Add nine milliliters of RPMI supplemented with 10%FCS to each tube, then centrifuge the tubes at 450 G for five minutes at four degrees Celsius. Use a Pasteur pipette with a vacuum to aspirate the supernatant, taking care not to touch the cell pellet.Add three milliliters of 100%stock isotonic density gradient solution to the cell pellet, then add additional RPMI 10%FCS media to bring the final volume to 10 milliliters and resuspend the cell pellet. Invert and mix the tube well, then insert a serological pipette with one milliliter of 70%stock isotonic density gradient solution into the bottom of the tube. Slowly underlay the solution, being careful not to make bubbles.Remove the serological pipette from the tube, making sure not to disturb the gradient. Centrifuge the tube at 800 G for 30 minutes at four degrees Celsius, then aspirate the supernatant, including the myelin debris layer, until two to three milliliters remain in the tube. Use a one milliliter pipette to harvest the cell layer between the 30%and 70%density gradient and transfer it to a new 15 milliliter tube.Add RPMI 10%FCS media to bring the final volume to 15 milliliters, then centrifuge the cells at 450 G for five minutes at four degrees Celsius. This protocol was used to assess B and T cells in the meninges, brain, and spinal cord during progressive multiple sclerosis. Gating was conducted to distinguish CD45 high peripherally derived infiltrating immune cells from CD45 low microglia and CD45 negative neurons, astrocytes, and oligodendrocytes.Few CD45 high cells were present in the spinal cord and brain tissue of sham-treated mice. The same gating cutoff for CD45 high expression was used in the meninges to identify CD45 high immune cells and exclude non-immune cells. In all TMEV-IDD CNS tissues, increased percentages of CD45 high immune cells were observed compared to sham treated mice.During chronic TMEV-IDD, the percentage of B cells among CD45 high immune cells in the brain and spinal cord was higher compared to the meningeal compartment. Decalcified brains and spinal cords were imaged to identify the localization of B and T cells within the CNS compartment. Conventional extraction of the brain from the skull cap resulted in an intact pia layer, but the remaining meningeal layers were excluded.In both decalcified brains and spinal cords, all meningeal layers were intact. Isotype switched B cells and T cells were localized with IgG and CD3 expression, respectively. Costaining IgG with ER-TR7 revealed that B cells were present in the CNS parenchyma and the meninges, while cellular aggregates within ER-TR7 positive meninges contained multiple IgG positive B cells and CD3 positive T cells.A critical step in these protocols is the careful extraction of the CNS tissues, essential for improving the purity of single cell suspensions and for obtaining histology tissue with high quality morphology. The described techniques allow for the comprehensive analysis of cells occupying the CNS compartment, ideal for examining cell phenotype, function, and localization under homeostasis and during disease pathogenesis.

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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 ...