NS088206和ES026892:这项工作是由健康（NIH）全国学院资助。在尤金和琳达·劳埃德主席赋予给AGK和院长教授以AK也被确认。

动物和协议的程序使用批准和机构动物护理和使用委员会（IACUC）在爱荷华州立大学（埃姆斯，IA，USA）的监督 1.混合胶质细胞培养物的种植<ol><li>迅速斩首1-2日龄幼仔用5.5英寸操作剪刀，并立即放置在头冰上的50毫升管中。请注意，这是断头安乐死的方式。 </li><li> 在层流气流罩，使在颅骨和使用4.5英寸直微解剖剪脑膜一个小切口。开始从尾端切割喙端（鼻）。通过使用由断头形成的开口获取的皮肤下面。 <ol><li>切口后，剥离半球一侧的一个。然后使用一对弯曲的或钩状镊子的删除整个大脑。 </li></ol></li><li>沉浸大脑（一个或多个）在含有0.25％胰蛋白酶ethylenediaminetetraacet新50mL试管在37℃水浴中吃15分钟乙酸（EDTA）。使用2毫升每脑胰蛋白酶-EDTA的。 </li><li>通过添加和删除的新鲜生长培养基（10％FBS，DMEM / F12，1％青霉素/链霉素，1％L-谷氨酰胺，1％丙酮酸钠和1％非必需氨基酸）洗涤脑（S）媒体。重复此4倍。 </li><li>对于每个脑，板含有生长培养基两个T-75烧瓶中。因此，每个脑加入2毫升的生长培养基到管上。因此，这将是1毫升脑匀浆中的与8-9毫升，每T-75烧瓶中的生长培养基的等效。 </li><li> 通过研磨脑（或多个）具有不同开口尺寸的移液管，以便从最大到最小均化。当它是可见的大脑组织没有变小，过渡到下一个吸管。在研磨结束时，悬挂应该是明确的，没有可见的块。 <ol><li>使用一个25毫升移液管，5毫升吸管，然后毫升吸管顺序10中。 </li></ol></li><li>通过每个homog通过70微米的细胞滤网enous脑悬浮液，使之成为一个单一的细胞培养物。 </li><li>对于每个匀化脑，包含板两个T-75烧瓶生长培养基中，在步骤1.5（均化脑的1毫升8-9毫升，每T-75烧瓶中的生长培养基）中所述。 </li><li>转变增长媒体6 d后，成长，直到第 16天的隔离。 </li></ol> 2.小​​胶质细胞的分离<ol><li> 后16天，从烧瓶中除去生长培养基，并放置在新的50ml管中。加入3毫升0.25％胰蛋白酶-EDTA的每个T-75烧瓶中。摇定轨振荡器上在室温下5分钟的烧瓶中。 <ol><li>离心机在0.4×g离心5分钟，除去的生长培养基，并使用停止在随后的步骤中胰蛋白酶-EDTA反应。 </li></ol></li><li>振摇5分钟后，添加至少4毫升生长培养基（新鲜的或从2.1.1所使用的介质）中的以停止胰蛋白酶-EDTA反应。 </li><li>磨碎，以确保所有的C厄尔已经分离。 </li><li>研磨后，通过将细胞通过70微米的细胞滤网，以使之成为一个单一的细胞培养物，进行细胞计数，然后在0.4×g下降速细胞5分钟。 </li><li>对于每100×10 6个细胞（大约15×T-75烧瓶中），可以使用1毫升推荐的介质（2％FBS，DPBS（钙和镁的氯化物免费），1mM EDTA）中的重悬细胞沉淀。 注:所有下列步骤专为1层毫升分离。 </li><li>取5毫升聚苯乙烯管中，加入推荐的介质的1毫升，并标记弯液面。添加推荐的介质为2.5毫升和标记这一点。丢弃所推荐的媒体和重新悬浮的细胞转移到5毫升聚苯乙烯管。 </li><li>添加大鼠血清50μL的每1毫升悬浮细胞的。在室温下孵育5分钟。 </li><li>通过混合组分A的25μL和组分B的25μL这些组件是专有的制备选择鸡尾酒。 </li><li>添加501，L选择鸡尾酒与细胞的。在室温下孵育5分钟。 注:对于更纯粹的文化，重复步骤2.9（建议，但不是强制性的）。 </li><li>涡街球为45秒。添加80μL每1mL样品的微球。在室温下孵育3分钟。 </li><li>通过将推荐的介质使体积到在聚苯乙烯管2.5毫升。 </li><li>放管中的磁体，在室温下3分钟。调整孵化，以增加纯度。慢慢倾出推荐的介质到15mL管与磁体仍然在聚苯乙烯管中。 </li><li>重复步骤2.12三次。附加磁性孵育可以被执行以增加纯度。 </li><li>加入3毫升的生长培养基和计数使用细胞计数器细胞的数目。 </li><li>板的细胞在相应聚-D-赖氨酸（PDL）的治疗中包被的平板上。处理细胞接种inPDL包被的板上之后48个小时。这允许细胞从分离的压力中恢复。 注:车使用完蛋免疫细胞化学如前面15所描述的培养物的纯度。 </li><li>板中的负部分（在一个15毫升管中收集），其大多含有星形胶质细胞，在生长培养基的T-75烧瓶中。 </li><li>在37℃培养箱中温育至少6个小时后，改变培养基，并让它生长O / N。拆分星形胶质细胞，第二天的治疗。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

旧版小胶质细胞的分离方法具有有限的回收率关于各种蛋白通过Western印迹分析和RNA通过qRT-PCR分析是不恰当的。差分的粘附性和温和的胰蛋白酶消化的方法是具有低的小神经胶质收率13，14，15两种常用的方法。基于列的CD11b的方法也有低回收率，但实现了比差的粘附性和温和的胰蛋白酶消化13，14，15，16，17更高的纯度。我们最初的无柱的CD11b的方法极大地提高了的分离产率，使其适合于蛋白质和RNA如Western印迹和qRT-PCR分析这样，但在成本纯度15下降。 我们的新修改的方法保留原始方法的产率和从〜95-97％至〜&gt; 99％稍微增加纯度，但在完成时间的一半，这对于细胞存活至关重要。在协议中的关键步骤可确保最佳的小胶质细胞的隔离。虽然斩首幼仔，应注意保持在冰面上的头和5-10分钟内，在层流室解剖。长时间大脑切除倍会使，因为它开始在环境温度下失去其坚固性很难得到完整的大脑，损害小胶质细胞的产量。值得注意的是，如果步骤2.9重复至少一次，存在在纯度和存活力可观察到的增加。如果从分离产率是低的，该分离可以使用每推荐的媒体毫升更少烧瓶，或通过增加分离的体积（&gt; 1毫升）重复。降低细胞拥挤通常将提高纯度，生存力，和产量。磁性孵育时间可在步骤2.12进行扩展以增加纯度。这个可以确保适当的磁性附加到小胶质细胞微球的结合。此外，附加的磁性孵育可以在步骤2.9以增加纯度和产率的缘故进行。 该协议的可行性是等于或大于原始的CD11b方法更好;然而，新的方法是精短。从该精制方法得到的另一个显著优点是利用用于荧光显微术，这是由PE在原来的方法（ 表1）15所占据的红色通道的选择。虽然我们获得与方法小胶质细胞的高产率和纯度，由此分离获得的星形细胞产量是不太纯，因为它保留了一些成纤维细胞。如在步骤中描述2.17，电镀的星形胶质细胞和温育6小时后，将生长培养基应当用新鲜培养基替换。星形胶质细胞是第一个连接到瓶，而大部分的成纤维细胞留在停赛ension。更换媒体通常会除去大部分污染的成纤维细胞。尽管这种技术保证星形胶质细胞的高纯度，二次纯化方法来实现较纯的培养物是必要的。 总体而言，该修改的CD11b隔离方法生成在很短的时间量高纯度的初级小胶质细胞和星形胶质细胞。较短的隔离时间通常可以提高细胞的存活率。它提供了一个有用的模型系统，用于阐明既小胶质细胞和星形胶质细胞生物学的基础的信号传导机制。

小胶质细胞是中胚层起源的MYB-亚独立定居巨噬细胞，其选自c-kit + / CD45 -分化erythromyeloid在卵黄囊1，2的血岛祖细胞。一旦胚胎的小胶质细胞已定植的中枢神经系统（CNS），它们从一个变形虫过渡到网状形式3。因为它们的动态后果探测潜在的侮辱4良性脑实质这些成人小胶质细胞被列为监视者。虽然小胶质细胞只向CNS细胞群的约10％，其以瓦能力彼此之间确保了实质4,5的最大扫描。危险相关的分子模式（DAMP的），如α突触核蛋白6,7和淀粉样蛋白β8，或对athogen相关分子模式（PAMP），如脂多糖（LPS）9，经典激活的小胶质细胞，以促进其特征在于，回复到变形虫活动状态，产生一氧化氮，肿瘤坏死因子α（TNFα），白介素1β的炎症反应（IL-1β），IL-6，IL-12，和趋化因子CC基序配体2 9，10，11。在神经炎性病症，例如帕金森氏病，其中致病α突触核蛋白积累了，神经变性周期从多巴胺能神经元，其中释放出更多的聚集α突触核蛋白的死亡创建的，进一步促进小胶质细胞7的经典活化。类似于周巨噬细胞，小神经胶质细胞还可具有在抗炎细胞因子IL-4和IL-10的存在下交替地激活的能力，给他们的有效的促进神经修复和衰减炎症2，11 IAL。除了在中枢神经系统的免疫作用，小胶质细胞已被开发过程中修剪突触描述为神经回路的重要调节器。例如，Cx3cr1- KO小鼠具有较少致密的小胶质细胞和减少突触修剪，这导致树突棘，未成熟的突触，并不发达CNS 12的电图案的过剩。在CNS中的稳态了解这些生理复杂性和小胶质细胞的各种功能性作用是对寻求治疗剂靶向神经变性疾病的关键。 在神经免疫学领域， 在体外实验是因为机理研究更大的可行性非常可取的，较低的维护成本，并为是少时间和劳动密集。 Furthermo再，以分离细胞群的能力是在规定条件下以描绘那些靶细胞的功能性是至关重要的。无数小胶质细胞的分离方法存在，但它们可通过它们，以获得相对高的数量和纯度为广泛的实验13，14，15的能力的限制。例如，分化11b的簇（细胞CD11b）是单核细胞，巨噬细胞，小胶质细胞和16的共同的表面标志物。通过利用细胞CD11b，磁分离的方法最初被描述为每新生儿脑17产生了基于列的办法〜99.5％的纯度，和〜1.6×10 6的小胶质细胞。我们实验室最近开发了一种无柱的CD11b磁分离方法15，我们在聚苯乙烯管通过用缀合藻红蛋白（PE）的单克隆抗体标记的CD11b进行。一种双特异性第二ARY抗体到PE和与PE葡聚糖复合物。一旦结合，葡聚糖包被的磁性颗粒被引入，其结合所述抗体复合物的葡聚糖端。最后，聚苯乙烯管放置在小神经胶质细胞隔离的磁铁。这种方法增加了一倍的产率每〜新生儿脑，但在减少纯度〜97％的成本3.2×10 6的小胶质细胞。 在本文中，我们展示了一个快速和精制无柱的CD11b磁选协议（ 图1）。由于CD11b的磁分离试剂盒的价格是一样的这种改进的方法仍然是我们原来的无柱的方法是可行的。完成时间减半，这可以最大限度地提高细胞的存活和产量至关重要的减少。值得注意的是，从这个优化的方法实现纯度〜&gt; 99％，比从我们的实验室15开发的原始无柱方法实现的纯度显着改善。最重要的是，细胞CD11b-PE没有被利用，从而无需从光孵育远，并允许荧光显微镜使用红色通道的。最后，如在原来的CD11b的方法，用这种改进的方法获得高产率和高纯度的星形细胞级分。星形胶质细胞是中枢神经系统中最大量的胶质细胞，导致的想法，他们的自我平衡的功能有关的病理生理学18不可缺少的。这些胶质细胞在不同的生理功能，如形成血 - 脑屏障，提供营养支持，维持神经递质的动态平衡，响应损伤神经保护作用形成胶质疤痕，学习和记忆，和神经炎症，体现他们的调查潜在的胶质中发挥作用生物学19。形态与小胶质细胞和星形胶质细胞的功能都通过激光共聚焦显微镜，免疫印迹，实时定量聚合酶链反应（定量RT-PCR），G被确定里斯亚硝酸盐测定中，和Luminex多重细胞因子测定。由这个协议所提供的精细化提供更高的置信度涉及小胶质星形细胞或纯度，与红色信道的可用性荧光显微镜的更广泛的应用，并节省了时间，所有这些都是在体外实验重要。

<table>
	<tbody>
		<tr>
			<td>EasySep CD11b Separation Kit II</td>
			<td>StemCell Technologies</td>
			<td>18970</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1x)</td>
			<td>Life Technologies&#160;</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies&#160;</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>13H469</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco by Life Technologies</td>
			<td>25200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Gibco by Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and refined cd11b magnetic isolation of primary microglia with enhanced purity and versatility

小胶质细胞是主要的反应，以中枢神经系统损伤;然而，还有许多关于他们在调节神经炎症的作用尚不清楚。小胶质细胞是功能类似于在巨噬细胞炎性测量应力中胚层细胞。经典的（M1型）和替代（M2型）的巨噬细胞的激活也已经扩展到小神经胶质细胞中是为了更好地理解下面的相互作用这些表型具有在神经炎性病症​​，例如帕金森氏，阿尔茨海默氏症，和亨廷顿氏病。 体外实验利用初级小胶质细胞提供了可以扩展到体内环境的快速和可靠的结果。虽然这是在体内实验具有明显的优势，隔离的小胶质细胞而实现最佳的纯净，足够的产量一直是个挑战。目前使用的常用方法无论是从回收率低，纯度低，或两者受苦。在此，我们高程模型onstrate该实现中的时间量的一半高的细胞回收和提高的纯度的无柱的CD11b磁分离方法的改进。我们建议这种方法优化为主的小胶质细胞分离为研究神经炎症和神经退行性病变的目的非常有用的模型。

小神经胶质细胞使用细胞CD11b阳性选择试剂盒分离II具有高纯度原代小鼠的小胶质细胞使用上述方案分离并接种于聚-D-赖氨酸包被的盖玻片上，以检查隔离的纯度。每孔和免疫细胞化学分析铺板万个细胞使用离子化的钙结合适配器分子1（Iba1）作为小胶质细胞和神经胶质纤维酸性蛋白（GFAP）作为星形胶质细胞的标记物的标记物以检查所述分离的小胶质细胞的纯度进行。离体培养中表达只无Iba1 GFAP的表达（ 图2A），表明培养物中分离是纯的小胶质细胞。不同于初级小胶质细胞的分离，取得纯度〜97％其它先前公布的方法，使用这种方法，我们在一半的时间得到的〜&gt; 99％纯度。为了进一步验证日的纯度<html>是文化，我们进行了印迹20 Iba1和GFAP。免疫印迹分析进一步揭示了从混合胶质细胞培养这种隔离是几乎纯的小胶质细胞培养物（ 图2B）。 修改后的分离过程中没有从磁珠任何自体荧光使用用于小胶质分离先前CD11b的分离试剂盒时由Gordon 等人提到的红色通道不能用于进行免疫细胞化学（ICC）。 15因为在分离过程中使用PE标记的。使用这种新的游离PE-分离试剂盒，也可以使用用于免疫细胞化学分析（ 图3）红色通道。从这个ICC，显而易见的是，这种新方法使我们能够使用流式细胞仪或其他荧光成像研究中的所有通道。 ve_step" FO:保持-together.within页= "1"&gt;隔离小胶质细胞培养功能到LPS刺激的反应要验证分离的小胶质细胞是功能活性，我们处理的细胞用LPS，一种广泛使用的兴奋剂9探测关于各种促炎因子前激活的小胶质细胞，24个小时。经典小胶质细胞活化伴随着释放的亚硝酸盐和各种促炎性细胞因子进入媒体。因此，我们使用多个互补测定法来证实我们的分离的小胶质细胞的功能活性。首先，我们用Griess检测表明LPS诱导的显着分泌亚硝酸盐从分离的小胶质细胞（ 图4A）。为了进一步验证分离的小胶质细胞的活性，我们使用定量RT-PCR，以显示（信使核糖核酸）NOS2，小神经胶质细胞的炎症的另一个标志的mRNA水平，显著增强无线个LPS处理（ 图4B）。接下来，我们使用了基于珠粒的多重测定，的Luminex 21，以验证LPS刺激显著促炎性细胞因子的分泌从培养小胶质（ 图5A）。此外，我们通过定量RT-PCR分析证实，LPS处理几个增强促炎细胞因子，包括IL-1β和TNFα（ 图5B）的基因表达水平。所有这些数据一起表明，原代小胶质使用这种新精制方法分离是有功能活性的，并显示出类似的活化轮廓初级小胶质细胞使用先前公开的方法分离的。 孤立的小胶质细胞可用于信令研究使用小胶质细胞隔离以前的方法，运行西方BLO吨用于信令的研究是困难的和不可行由于低收率和纯度。以前，我们已经表明，的Fyn，Src家族激酶，参与促炎信号级联在小神经胶质细胞9。在这里，我们镀百万小神经胶质细胞在12孔板中，并在48个小时后，收集细胞以用于本机的Fyn运行Western印迹和Src激酶在酪氨酸残基416（P-SRC-Y416）磷酸化。我们能够在我们的孤立的小胶质细胞（ 图6），同时检测菲英岛和P-SRC-Y416水平，表明该方法适用于信令研究Western印迹技术。 来自小胶质细胞分离负馏分含有星形胶质细胞可用于信令研究星形胶质细胞是神经炎症的主要参与者，并了解背后的而aStr信号机制ocytic炎症和神经元的星形胶质细胞的串扰是非常重要的19。在这里，我们表明，从小胶质分离负馏分含有GFAP阳性星形细胞的细胞（ 图7A）。此外，我们的用于的Fyn和对SRC-Y416（ 图7B）的Western印迹分析表明，这两种蛋白质的可以在阴性级分，以及被检测到。这些结果一起表明，该负分数是用于目的在于鉴定蛋白质的炎症性信号传导级联重要星形细胞研究的理想制剂。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55364/55364fig1.jpg" /> 图1:从1-2天-产后幼鼠小胶质细胞的分离的示意图 。 <a href="http://ecsource.jove.com/files/ftp_upload/55364/55364fig1large.jpg" target="_blank">请点击此处查看大图</a>这一数字。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55364/55364fig2.jpg" /> 图2:小胶质细胞隔离用的CD11b阳性选择试剂盒II的纯度高。 （A）分离的小胶质细胞培养物的免疫细胞化学在绿色通道红色通道和IBA1探查GFAP。分离的小胶质细胞培养物探查GFAP（〜51 kDa的），IBA1（〜15kDa的）和β肌动蛋白（〜42kDa的）的（B）免疫印迹。比例尺=20μm以下。 <a href="http://ecsource.jove.com/files/ftp_upload/55364/55364fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55364/55364fig3.jpg" /> 图3:修改后的分离过程没有从磁珠任何自动荧光。 </stron克使用新的分离方法和旧的（原始）方法在红色通道绿色通道和PE荧光探查IBA1孤立的小神经胶质培养物&gt;免疫细胞化学。比例尺=30μm以下。 <a href="http://ecsource.jove.com/files/ftp_upload/55364/55364fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55364/55364fig4.jpg" /> 图4:LPS刺激增加亚硝酸盐产生在小神经胶质培养。 （A）分离的小神经胶质细胞用1μg/ mL的LPS处理24个小时，并处理介质收集，以确定由Griess检测亚硝酸盐的释放。 （B）Q-RT-PCR用于从NOS2用LPS处理24小时分离的小胶质细胞。的数字代表从2个或更多独立实验的平均值±SE。使用学生t检验（分析数据* P0; 0.05，** P &lt;0.01，*** P &lt;0.001）。 <a href="http://ecsource.jove.com/files/ftp_upload/55364/55364fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55364/55364fig5.jpg" /> 图5:小胶质细胞LPS诱导的促炎细胞因子释放。 （A）Luminex多重测定从与1μg/ mL的LPS处理24小时分离的小神经胶质细胞收集处理介质上进行。 （B）Q-RT-PCR对IL-1β和TNFα从用LPS处理24小时分离的小胶质细胞。的数字代表从2个或更多独立实验的平均值±SE。使用学生t检验分析数据（* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001）。 <a href="http://ecsource.jove.com/files/ftp_upload/55364/55364fig5large.jpg" target="_blank">请点击此处</a><html>查看该图的放大版本。 <img alt="图6" class="xfigimg" src="/files/ftp_upload/55364/55364fig6.jpg" /> 图6:隔离小胶质细胞可用于研究信令。 Western blot分析表明，菲英岛和P-SRC-Y416可以从我们的新完善的方法分离出的小胶质细胞进行检测。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55364/55364fig7.jpg" /> 图7:来自小胶质细胞分离的消极部分包含星形胶质细胞可用于信令研究。 （A）蛋白质印迹法显示，负级分含有GFAP阳性细胞。 （B）蛋白质印迹分析显示，的Fyn和对SRC-Y416可从GFAP阳性细胞进行检测。目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> 目前的方法 </td><td colspan="2"> 修改方法 </td></tr><tr><td>成本</td><td> $ 620 </td><td colspan="2"> $ 620 </td></tr><tr><td>时间</td><td> 55分钟</td><td colspan="2"> 25分钟</td></tr><tr><td>荧光</td><td>是（红色通道） </td><td colspan="2">没有</td></tr><tr><td>纯度</td><td> 〜97％ </td><td colspan="2"> 〜99％ </td></tr></tbody></table> 表1:小神经胶质精制方法分离和隔离的原来的方法之间的比较分析。

Watch this Scientific Journal Video about Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility at JoVE.com

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

这里，我们提出用于体外实验的协议来隔离来自出生后小鼠幼仔的小胶质细胞（1天）。此简易分离方法产生了高收率和纯度，在替代方法一个优点显著允许阐明小胶质生物学的目的范围广泛的实验。

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

9.7K 次观看.  Iowa State University. 这里，我们提出用于体外实验的协议来隔离来自出生后小鼠幼仔的小胶质细胞（1天）。此简易分离方法产生了高收率和纯度，在替代方法一个优点显著允许阐明小胶质生物学的目的范围广泛的实验。 

视频: Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

科研

神经科学

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...