我们感谢乔治华盛顿大学基因组学核心的 Castle Raley 进行 RNA 分析，并感谢 Q2 Lab Solutions 的 RNA 测序分析。这项工作得到了美国国家神经疾病和中风研究所[资助号F31NS117085]和Vivian Gill研究基金会对Robert H. Miller博士的支持。 图 1 是使用 BioRender.com 创建的。

所有动物护理和实验程序均在乔治华盛顿大学医学与健康科学学院（美国华盛顿特区;IACUC#2021-004）。该研究利用了10周至5个月的雄性和雌性C57BL / 6J野生型（WT）小鼠，这些小鼠来自商业供应商（见 材料表）并安置在乔治华盛顿大学。协议工作流程的概述如 图 1 所示。
1.脊髓的准备
<ol><li>使用Avertin（12.5mg / mL的2,2,2-三溴乙醇和2.5%2-甲基-2-丁醇在无菌水中）麻醉动物（见 材料表）。确认小鼠对脚趾和尾巴捏合没有反应。</li>
	<li>用冷 1x Dulbecco 磷酸盐缓冲盐水 （DPBS） 进行心脏灌注，以去除外周血单核细胞。<ol><li>将动物放在浅托盘上，并做一个侧切口以暴露胸膜腔。将连接到蠕动泵的 23 G 灌注针（参见 材料表）插入左心室并激活泵。一旦冷 DPBS 流经心脏，在右心房创建一个小切口。 注意:在此步骤中首选更快的灌注速率以增强细胞活力。血液应在 1 分钟内冲洗干净。清除肝脏提示灌注成功。</li>
	</ol></li>
	<li>用大剪刀将动物斩首并移除脊柱14.将其浸没在含有 Ca2+/Mg2+/0.2% 葡萄糖的冷 1x DPBS 中，放在冰上的培养皿中进行组织冲洗。</li>
	<li>从现在开始，确保所有步骤都在无菌环境中进行。使用液压挤压14 隔离整个脊髓。使用一根 18 G 针头和一个 10 mL 注射器，注射器中装满含有 Ca2+/Mg2+/0.2% 葡萄糖的冷 1x DPBS。将脊髓转移到放置在冰袋上的培养皿中，冰袋中含有足够的冷 1x DPBS，含 Ca2+/Mg2+/0.2% 葡萄糖以淹没整个组织。</li>
	<li>在层流罩内的显微镜下小心地去除脑膜。将脊髓转移到冰袋上的空培养皿中，并使用10号刀片手术刀将整个脊髓切成2-3毫米的部分。</li>
</ol>2. 酶促细胞解离
<ol><li>按照制造商的方案，从市售的成人脑解离试剂盒中加入酶混合物 1 和酶混合物 2（参见 材料表）。将培养皿中的酶旋转数次以确保脊髓的均匀涂层，然后在37°C下孵育30分钟。每 5 分钟轻轻旋转培养皿，以防止组织结块并促进试剂均匀分布。</li>
	<li>从培养箱中取出培养皿，在最小必需培养基（MEM）中加入350μL0.46mg/mL DNAse I（参见 材料表）。轻轻旋转混合。</li>
	<li>加入 1 mL 冷 DMEM/0.5% 胎牛血清 （FBS），轻轻旋转混合。立即将全部内容物转移到 5 mL 试管中。</li>
</ol>3.机械细胞解离
<ol><li>使用 P1000 移液管，轻轻研磨组织三次，确保每次都抽出组织碎片以进一步解离组织。然后，使用塞住的 9 英寸玻璃巴斯德移液管，再轻轻研磨五次，确保在此过程中不会产生气泡。</li>
	<li>将 5 mL 试管直立放置，让组织在底部沉降 30 秒。组织沉降后，使用 P1000 移液管吸取上清液，并将其通过 30 μm 过滤器放入 15 mL 锥形管中。</li>
	<li>在相同的 5 mL 试管中加入 1 mL 冷 DMEM/0.5% FBS。使用巴斯德移液管，轻轻研磨三次。</li>
	<li>让组织在底部沉降 30 秒，然后将上清液通过 30 μm 过滤器并将其收集到与之前相同的 15 mL 管中。再次重复步骤 3.3 和步骤 3.4。</li>
	<li>在室温（RT）下以300× g 离心过滤的细胞悬浮液10分钟。使用真空吸气器小心地除去并丢弃所得上清液。</li>
</ol>4. 髓鞘去除
<ol><li>按照制造商的方案，使用成人脑解离试剂盒中提供的碎屑清除溶液 （DRS） 去除髓鞘。去除碎片后，向细胞沉淀中加入 5 mL 冷 DMEM/0.5% FBS，并轻轻倒置试管 3 次。在室温（RT）下以300× g 离心细胞悬液10分钟。 注意:如果细胞将用于 体外 研究并将保留在培养物中，则应使用预热的DMEM / 0.5%FBS。</li>
	<li>弃去上清液并将细胞沉淀重悬于 1 mL DPBS/0.5% FBS 中（冷用于 RNA 研究，温用于 体外 测定）。使用台盼蓝对细胞进行计数并评估活力。 注意:来自多种动物的细胞悬液可以组合以增加细胞浓度。</li>
	<li>通过加入 DPBS/0.5% FBS 洗涤细胞，总体积高达 5 mL。在室温下以300× g 离心悬浮液10分钟，并用移液管弃去上清液。</li>
</ol>5.小胶质细胞和星形胶质细胞分离
<ol><li>按照制造商的方案用抗CD11b或抗ACSA-2微珠标记细胞（参见 材料表）。</li>
	<li>根据制造商的说明，使用MS色谱柱通过阳性选择对细胞进行排序（请参阅 材料表）。分选后，将分选的细胞以300× g 离心10分钟，并弃去上清液。</li>
</ol>6. 用于 体外 检测的铺板细胞
注意:如果细胞不会被接种并立即用于RNA分析，请继续执行步骤8。
<ol><li>将细胞重悬于 1 mL 温热 DMEM/0.5% FBS 中。使用血细胞计数器对细胞进行计数并评估其活力（参见 材料表）。</li>
	<li>将细胞浓度调节至每 50 μL 75,000 个细胞。 在 24 孔板中向每个 Poly-L-赖氨酸包被的盖玻片11 中加入 50 μL 细胞悬液。</li>
	<li>在37°C孵育2小时，使细胞粘附在盖玻片上。</li>
	<li>小心地向每个孔中加入 450 μL 温热的 DMEM/5% FBS/青霉素-链霉素（Pen-Strep，参见 材料表）。</li>
	<li>3 天后，用 250 μL 温热的 DMEM/5% FBS/Pen-Strep 替换一半的培养基。为了进行维护，每 4-5 天用 500 μL 温热的 DMEM/5% FBS/Pen-Strep 完全更换培养基。</li>
</ol>7. 免疫组化
注意:最好在至少 3 天后至少更换一次培养基后进行免疫组化分析。这确保了碎片已被清除，细胞已完全粘附在盖玻片上。
<ol><li>在室温（RT）或37°C下取出培养基，并用抗小鼠单克隆抗体O4（1:100）（参见 材料表）在温热的DMEM / 5% FBS / 10%正常山羊血清（NGS）中标记细胞15分钟。 注意:如果细胞不会用O4标记，请跳过此步骤并继续执行步骤7.3。</li>
	<li>将盖玻片浸入温热的DMEM / 5%FBS中三次，轻轻冲洗盖玻片。在温热的DMEM / 5% FBS / 10%NGS中加入山羊抗小鼠594IgM（1:300）（参见 材料表），并在室温下在黑暗中孵育15分钟。用温热的 DMEM/5% FBS 轻轻冲洗 3 次。</li>
	<li>用冷的5%乙酸/甲醇在4°C下固定细胞15分钟。 用 1x PBS 洗涤 3 次，然后用适当的抗体标记:抗兔 GFAP （1:500）、抗小鼠 GFAP （1:500） 或抗兔 Iba1 （1:500） 在 1% Triton-X100/10% NGS 的 1x DPBS 中（见 材料表）。在室温下孵育1小时。 注意: 如果盖玻片已经被 O4 染色，请将其放在黑暗中。</li>
	<li>用 PBS 轻轻冲洗 3 次。用适当的二抗标记:抗小鼠594 IgG（1:500），抗兔488 IgG（1:500）（参见 材料表）。在室温下在黑暗中孵育 30 分钟。</li>
	<li>用 PBS 轻轻冲洗 3 次。用DAPI（1:1000）在室温下复染1分钟。 用PBS冲洗三次，加入封固剂（参见 材料表），并将盖玻片安装到载玻片上。</li>
</ol>8. RNA提取
注意:理想情况下，至少应该有 100,000 个细胞来提取足够的 RNA 进行分析。如有必要，可以合并来自2-3个脊髓的细胞。
<ol><li>按照制造商的方案使用市售的RNA分离试剂盒提取RNA（参见 材料表）。对于使用试剂盒中提供的RW1洗涤缓冲液的每个洗涤步骤，将细胞留在洗涤缓冲液中再放置2分钟。</li>
	<li>根据制造商的说明，使用DNase I去除任何剩余的基因组DNA（参见 材料表）。在室温下与DNase孵育15分钟，然后用RW1缓冲液洗涤。</li>
	<li>为了提高RNA产量，在洗脱之前，将样品在室温下在无RNA酶的水中孵育10分钟。使用生物分析仪15 评估RNA的完整性和数量（参见 材料表）。</li>
</ol>

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

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没有

分离纯活的原代细胞对于研究特定细胞类型的结构和功能至关重要。在成年小鼠中，特别是在脊髓中，这项任务带来了重大挑战，因为现有的方案通常不是针对成年脊髓量身定制的10,17。该方案提供了一种高效且具有成本效益的方法，适用于各种下游应用，包括细胞培养、流式细胞术、组织学和转录组学研究。
脊髓制备的速度在确?...

星形胶质细胞和小胶质细胞是多功能神经胶质细胞，在中枢神经系统 （CNS） 中起着至关重要的作用，包括调节神经元功能、促进中枢神经系统发育、维持血脑屏障以及参与其他关键过程等职责 1,2,3,4.除了在维持体内平衡方面的作用外，这些神经胶质细胞在损伤和修复机制中也起着关键作用。小胶质细胞以其吞噬、炎症和损伤后的迁移能力而闻名 5,6,7。疾病中的星形胶质细胞反应同样多种多样，包括炎症、神经胶质瘢痕的形成和血脑屏障的损害 8,9。尽管我们对小胶质细胞和星形胶质细胞在中枢神经系统中的有害和修复作用的理解有所增长，但它们的结构和功能固有的异质性需要强大的工具来研究它们在各种情况下。
进一步了解小胶质细胞和星形胶质细胞在健康和疾病中的作用需要 体内 和 体外 研究的综合方法。 体内 技术利用神经胶质细胞和中枢神经系统内神经元之间错综复杂的串扰，而 体外 方法在评估特定刺激下的单细胞功能或反应时被证明是有价值的。每种方法都有独特的优势; 体外 研究对于了解这些细胞类型的特定作用至关重要，而无需来自邻近细胞的直接或间接输入。此外，利用永生细胞系的 体外 检测具有某些好处，包括无限增殖的能力、成本效益和易于维护。然而，重要的是要注意，与细胞系相比，原代细胞更接近地模仿正常的生理反应。这种生理相关性在功能测定和转录组学分析中至关重要。
获得原代细胞，特别是从成年小鼠脊髓中获取原代细胞的挑战之一在于样本的数量和活力。成人脊髓比大脑小，含有大量的髓鞘，这带来了独特的困难。虽然有几种已发表的方案详细说明了从新生动物或成年小鼠大脑中分离纯、活的神经胶质细胞10,11,12,13，但这些方法可能不适合研究脊髓特异性的疾病和损伤。在该方案中，我们提供了一个全面的程序，可以有效地从成年小鼠脊髓中分离纯、活的小胶质细胞和星形胶质细胞，促进细胞培养和转录组学分析的下游应用。该方案已成功用于从10周至5个月的成年小鼠中分离这些细胞，证明了其在各种情况下的实用性，包括涉及条件敲除小鼠的研究，药物反应，发育研究和年龄相关模型。

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			<td>2,2,2-Tribromoethanol</td>
			<td>Sigma Aldrich</td>
			<td>T48402</td>
			<td></td>
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		<tr>
			<td>24 well tissue culture plate</td>
			<td>Avantor</td>
			<td>10861-558</td>
			<td></td>
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			<td>2-Methyl-2-butanol, 98%</td>
			<td>Thermo Fisher</td>
			<td>A18304-0F</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>1:1000</td>
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			<td>45% glucose solution</td>
			<td>Corning</td>
			<td>25-037-CI</td>
			<td></td>
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			<td>5 mL capped tubes</td>
			<td>Eppendorf</td>
			<td>30122305</td>
			<td></td>
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			<td>Acetic acid</td>
			<td>Sigma-Adlrich</td>
			<td>A6283</td>
			<td></td>
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		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>103-107-677</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ACSA2 Microbead Kit</td>
			<td>Miltenyi</td>
			<td>130-097-679</td>
			<td></td>
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			<td>Anti-Iba1</td>
			<td>Wako</td>
			<td>019-1974</td>
			<td></td>
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		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild-type (WT) mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-093-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Celltrics 30 &#181;m filter</td>
			<td>Sysmex Partec</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chamber (Hemacytometer)</td>
			<td>Hausser Scientific Co</td>
			<td>3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>D4527-40KU</td>
			<td></td>
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		<tr>
			<td>Distilled water</td>
			<td>TMO</td>
			<td>15230001</td>
			<td></td>
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		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>11320074</td>
			<td></td>
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		<tr>
			<td>DNase for RNA purification</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
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		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Thermo Fisher</td>
			<td>14040117</td>
			<td></td>
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		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>A5209401</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody (mouse)</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>GFAP antibody (rabbit)</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse 594 IgG</td>
			<td>Invitrogen</td>
			<td>a11032</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse 594 IgM</td>
			<td>Invitrogen</td>
			<td>a21044</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit 488 IgG</td>
			<td>Invitrogen</td>
			<td>a11008</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Iba1 antibody (rabbit)</td>
			<td>Wako</td>
			<td>019-1974</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>MACS Separator</td>
			<td>Miltenyi</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex C/L Pump System</td>
			<td>Thermo Fisher</td>
			<td>77122-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>15-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Adlrich</td>
			<td>439193</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 Antibody</td>
			<td>R&#38;D</td>
			<td>MAB1326</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugged 9&#34; glass pasteur pipette</td>
			<td>VWR</td>
			<td>14672-412</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>74034</td>
			<td></td>
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		<tr>
			<td>Royal-tek Surgical scalpel blade no. 10</td>
			<td>Fisher scientific</td>
			<td>22-079-683</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Vein Infusion Set, 23 G x 19 mm</td>
			<td>Kawasumi</td>
			<td>D3K2-23G</td>
			<td></td>
		</tr>
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</table>

isolation of pure astrocytes and microglia from the adult mouse spinal cord for in vitro assays and transcriptomic studies

星形胶质细胞和小胶质细胞在中枢神经系统发育、损伤反应和神经退行性疾病中起着关键作用。这些高度动态的细胞表现出对环境变化的快速反应，并在形态、转录谱和功能方面表现出显着的异质性。虽然我们对神经胶质细胞在健康和疾病中的功能的理解已经有了很大的进步，但仍然需要在损伤或损伤的背景下进行 体外细胞特异性分析，以全面表征不同的细胞群。与细胞系或新生动物相比，从成年小鼠中分离细胞具有多项优势，因为它允许在病理条件下和特定时间点分析细胞。此外，专注于脊髓特异性分离（不包括脑部受累）可以研究脊髓病理学，包括实验性自身免疫性脑脊髓炎、脊髓损伤和肌萎缩侧索硬化症。该方案提供了一种从成年小鼠脊髓中分离星形胶质细胞和小胶质细胞的有效方法，有助于立即或将来的分析，并在功能，分子或蛋白质组学下游研究中具有潜在应用。

该协议中概述的方法能够从成年小鼠脊髓中分离纯和活的小胶质细胞和星形胶质细胞，促进各种下游应用，包括 体外 功能或组织学测定和RNA分析。
体 外 研究的成功分离将导致数天的细胞连续增殖。与从新生动物中分离的细胞相比，成体细胞的增殖速度较慢，并且在最初几天可能存在一些碎片。到 体外 4 天 （DIV） 时，细胞应基本清除碎片，大多数细胞?...

Watch this Scientific Journal Video about Isolation of Glial Cells from the Adult Mouse Spinal Cord at JoVE.com

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

该方案概述了从成年小鼠脊髓中分离纯化的星形胶质细胞和小胶质细胞，促进了后续应用，如RNA分析和细胞培养。它包括详细的细胞解离方法和程序，旨在提高分离细胞的质量和产量。

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

我们的研究旨在使用疾病的临床前模型来表征中枢神经系统中的特定细胞群，特别是脊髓。我们正试图确定这些细胞在病理条件下和不同时间点是如何受到影响的。分析细胞可能具有挑战性，尤其是在中枢神经系统中，因为大脑和脊髓中的细胞形成一个高度复杂的基质，并且所有东西都非常紧密地连接在一起，因此很难在不损坏系统的情况下分离这些细胞。我们的方案通过从富含髓鞘的小成年小鼠脊髓中有效地分离出活的小胶质细胞星形胶质细胞，解决了现有研究脊髓疾病的方法的局限性。这使得在下游分析中对脊髓相关疾病进行体外研究成为可能，填补了关键的研究空白。该协议具有多种优点，例如在病理条件下和特定时间点分析星形胶质细胞和小胶质细胞。它还特别专注于从成人脊髓中分离神经胶质细胞，不包括大脑受累。这使得研究人员能够在细胞水平上研究脊髓病理学。

isolation-pure-astrocytes-microglia-from-adult-mouse-spinal-cord-for

Research

JoVE Journal

Neuroscience

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

1.6K 次观看.  The George Washington University School of Medicine. 我们的研究旨在使用疾病的临床前模型来表征中枢神经系统中的特定细胞群，特别是脊髓。我们正试图确定这些细胞在病理条件下和不同时间点是如何受到影响的。分析细胞可能具有挑战性，尤其是在中枢神经系统中，因为大脑和脊髓中的细胞形成一个高度复杂的基质，并且所有东西都非常紧密地连接在一起，因此很难在不损坏系统的情况下分离这些细胞。我们的方案?...

视频: 作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

脊髓电二:外吸电极的制作

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

一个程序，植入脊柱商会纵

细胞动力学在小鼠脊髓双光子成像

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

脊髓神经元从新生小鼠中分离和培养

密度梯度离心法分离小鼠原发胶质细胞的研究

拉米内切手术和脊髓窗口植入小鼠

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

副本

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

脊髓电二:外吸电极的制作

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

一个程序，植入脊柱商会纵

细胞动力学在小鼠脊髓双光子成像

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

脊髓神经元从新生小鼠中分离和培养

密度梯度离心法分离小鼠原发胶质细胞的研究

拉米内切手术和脊髓窗口植入小鼠

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从胚胎脊髓连合神经的解剖和文化