这项工作得到了克里斯托弗和丹娜基金会国际脊髓损伤研究联合会的支持, dr 和谢尔顿 g. 阿德尔森医学研究基金会, JPB 基金会, 诺华基础研究所,文森特和斯特拉的慷慨贡献, 和达蒙罗扬癌症研究基金会 (DRG-2125-12)。

所有涉及啮齿类动物的程序都符合斯坦福大学的指导方针, 遵守国家和州的法律和政策。所有动物程序都是由斯坦福大学的实验室动物护理管理小组批准的。
注意: 所有解决方案和缓冲区组合都在材料表中提供。
1. 为 CD11b Immunopanning 准备一个培养皿 (0 天)
注: 为每 1-2 只幼鼠大脑准备 1 immunopanning 菜。
<ol><li>在15厘米培养皿中加入25毫升50毫米的三 pH 9.5 溶液。</li>
	<li>添加山羊抗鼠 IgG (H + L 链) 到菜的最后浓度为6µg/毫升。涡流板均匀分布。</li>
	<li>在37摄氏度孵化 1-3 小时的菜肴。</li>
	<li>用带有 Ca2 +和 Mg2 +) 的 DPBS++ (磷酸盐缓冲盐水 (PBS)) 冲洗盘子三次, 然后用包含1µg/mL OX42 抗体的平移缓冲解决方案替换。把盘子在室温下放在平坦的表面上过夜。</li>
</ol>2. 组织收集 (1 天)
注: 该协议应采取〜 3-4 小时。
<ol><li>在开始之前, 确保所有的解决方案是无菌和冰冷却。冷却所有仪器在冰和消毒用乙醇在使用之前。</li>
	<li>遵循适当的监管程序, 用二氧化碳窒息的方式牺牲一只少年实验鼠。 注: 或者, 年轻的动物可能会牺牲的致命剂量氯胺酮/甲苯噻嗪 (100-200 µL 24 毫克/毫升氯胺酮, 2.4 毫克/毫升甲苯噻嗪)。如果动物不到14天的年龄, 必须使用氯胺酮/甲苯噻嗪。如果使用多种动物, 从一种动物中提取组织, 并将其放到冰前的 DPBS++上, 然后再进入随后的动物。</li>
	<li>捏一 hindpaw, 并确保完整的对停止响应从动物之前继续。</li>
	<li>Transcardially 灌注的动物与 10-30 毫升冰冷灌注缓冲使用 27½-G 针, 直到缓冲区运行清楚。 注: 灌注缓冲量根据动物的大小/年龄而定。</li>
	<li>灌注后立即取出动物的头。从脊髓中切开每一侧的枕髁用解剖剪刀, 小心不要损伤大脑。仔细切开每一侧后, 沿顶叶和前额骨切开一侧, 朝向鼻骨。用镊子仔细拉回头骨的顶端, 迅速删除大脑 (或中枢神经系统结构的兴趣), 并放置在冰上的预冷 DPBS++ 。</li>
	<li>对所有剩余的动物重复。 注: 在适当的无菌条件下, 所有步骤都应在层流罩内进行。</li>
</ol>3. 机械离解 (1 天)
<ol><li>在所有的大脑被收集, 把一个大脑切成1毫米3块, 在一个在冰上的培养皿与冷手术刀刀片, 并转移到冰冷的 dounce 均质机与 5-7 毫升冰冷 douncing 缓冲。一次分离一个大脑。</li>
	<li>使用 10-20 的温和和不完全的中风与松散拟合的 dounce 均质体分离组织。注意不要直接粉碎了均质机底部的组织, 而是通过活塞和均质机两侧的空间来推动组织。</li>
	<li>小心地卸下活塞, 防止气泡的引入。允许分离不良的组织块沉淀到均质体的底部, 并将上清转移到新的冷50毫升锥形管上。</li>
	<li>用新鲜的 douncing 缓冲区替换已删除的卷, 并重复步骤3.2 和 3.3, 总共 3-4 回合, 或直到所有组织都已分离。对每个大脑重复这个过程。</li>
</ol>4. 髓鞘摘除 (1 天)
注意: 髓鞘去除是用来隔离小胶质细胞从年龄比 P12 的动物。
<ol><li>用25毫升吸管测量50毫升圆锥管中细胞悬浮液的体积, 然后加入冰冷 douncing 缓冲器, 将总容积调整到33.5 毫升。</li>
	<li>在细胞悬浮液中加入10毫升髓鞘分离缓冲液 (MSB), 并通过多次反转管彻底混合。这将导致23% 最终浓度的 MSB 在43.5 毫升的体积。</li>
	<li>离心细胞为15分钟在 500 x g 在4°c 与缓慢的刹车。 注: 离心机应采取大约 1.5-2 分钟减速。这将产生一个上层的髓鞘和死细胞碎片, 有点阴暗的上清, 和一个小颗粒, 丰富的活细胞。</li>
	<li>用吸管去除髓鞘/碎片和上清上层。卸下顶层时要小心, 以确保尽可能多地删除它。</li>
	<li>并用重悬12毫升的平移缓冲器中的细胞颗粒。轻轻地 triturate 细胞悬浮, 以打破任何可能留下的细胞团簇。</li>
</ol>5. Immunopanning (1 天)
<ol><li>通过70µm 细胞过滤器通过细胞悬浮物来去除大的碎片或细胞团簇。</li>
	<li>用 DPBS++冲洗 OX42-coated 平移盘三次。不要让盘子在洗涤之间完全干燥。</li>
	<li>倒掉最后一个 DPBS++清洗并将过滤后的单元格悬挂应用于平移盘。轻轻地旋转盘子以分配细胞, 然后在室温下在平坦的表面上孵化出20分钟的平板以允许细胞黏附。不要孵育超过20分钟或细胞将变得很难从菜中恢复。</li>
	<li>用 DPBS++ 10 次冲洗平移盘以移除非粘附单元格。小胶质细胞将牢固地附着在盘子上, 所以每冲洗一遍盘子, 以确保去除其他非粘附单元。</li>
	<li>将最后一个 DPBS++清洗并替换为15毫升 DPBS++和 200 ul 胰蛋白酶 (1.25% 个库存解决方案)。</li>
	<li>孵化的菜为≤10分钟在37°c 与 10% CO2胰蛋白酶处理。 注: 不要持续长于10分钟或小胶质细胞将变得难以去除。</li>
	<li>trypsinization 10 分钟后, 小胶质细胞仍会粘在盘子里。倒入胰蛋白酶/DPBS++ , 用 DPBS++轻轻地冲洗 2x, 以除去胰蛋白酶, 代之以12毫升的冰冷小胶质细胞生长培养基 (米高梅)。</li>
	<li>将平移盘放在冰上2分钟, 以帮助削弱细胞/基质的相互作用, 并确保盘子是平的, 以防止平移盘的区域干燥。</li>
	<li>用10毫升的吸管和吸管控制器在高速上强力吸管, 从平移盘中恢复细胞。绘制一个 16 x 16 网格与流的液体从吸管, 试图删除所有的细胞。</li>
	<li>检查显微镜下的细胞在20X 倍放大, 以确保细胞已脱离板块。在盘子的顶端标记斑点, 细胞仍然卡住, 在这些区域重复吹打。</li>
	<li>收集细胞悬浮液和整除 3-4 毫升上清每15毫升圆锥管。旋转15分钟在 500 x g 在4°c 与缓慢的刹车。 注: 小胶质细胞通过少量的体积, 可以最大限度地恢复电池。</li>
	<li>吸入上清液, 将0.5 毫升米高梅与细胞颗粒一起留下。</li>
	<li>并用重悬在剩下的米高梅的每个颗粒, 并汇集所有的细胞从所有的管。</li>
	<li>用 hemocytometer 计数单元格。</li>
	<li>根据应用程序的步骤 6-7 中所述的板单元格。</li>
	<li>文化细胞在37°c 与 10% CO2。 注意: 单元格可以是最多 3-4 周的区域性, 并且有常规媒体更改, 或者没有媒体更改的一周。</li>
</ol>6. 斑点涂料组织培养板/盖玻片 (1 天)
<ol><li>板15µL 的胶原 IV 涂层直接在24良好的阴离子/阳离子涂层组织培养板的中心 (见材料表的细节) 和孵化15分钟在37°c 与 10% CO2。</li>
	<li>在米高梅 hemocytometer 稀释细胞到 2.3 x 105/毫升的细胞计数后。吸入胶原 IV 斑点, 并立即板15µL 细胞悬浮到此部位;这将提供 3.5 x 103单元格/点。孵育 5-10 分钟在37°c 与 10% CO2允许细胞坚持, 增加500µL CO2-平衡 TGF-β2/IL-34/cholesterol 包含生长媒介 (TIC) 温和地对井。</li>
	<li>如果镀在盖玻片, 第一涂层无菌玻璃盖玻片与10µg/毫升的策略 d-赖氨酸 (PDL)在 H 2 o 为 1 H 在 RT, 洗涤 3次与 H 2 O, 并且让干燥在敞篷在紫外光之下。一旦盖玻片干燥, 就按照步骤6.2 中所述, 对盖玻片进行现场电镀。</li>
</ol>7. 用于 RNA/蛋白质分离的电镀
<ol><li>在1天, 涂上24良好阴离子/阳离子涂层组织培养板的整个面积与胶原 IV 涂层和孵育10分钟-1 h 在37°c 与 10% CO2, 然后删除和立即板 3-5 x 104单元格/以及 CO2平衡TIC 媒体。</li>
	<li>在2天, 做好准备后的每一天, 为每个油井执行50% 媒体更改。死细胞往往聚集在井的中心, 所以从那里取媒体。</li>
	<li>每 2-3 天执行50% 媒体更改以维护区域性。如果媒体更改为区域性引入了不需要的变量, 则单元格可以在不发生任何媒体更改的情况下生存大约1周, 而不是3周的定期维护。</li>
</ol>

<ol>
	<li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Function+in+the+Central+Nervous+System+During+Health+and+Neurodegeneration.">Microglia Function in the Central Nervous System During Health and Neurodegeneration.</a> Annu Rev Immunol. 35, 441-468 (2017).</li><li>Wu, Y., Dissing-Olesen, L., MacVicar, B. A., Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Dynamic+Mediators+of+Synapse+Development+and+Plasticity.">Microglia: Dynamic Mediators of Synapse Development and Plasticity.</a> Trends Immunol. 36 (10), 605-613 (2015).</li><li>Lou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptor+P2RY12-dependent+microglial+closure+of+the+injured+blood-brain+barrier.">Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier.</a> Proc Natl Acad Sci U S A. 113 (4), 1074-1079 (2016).</li><li>Haynes, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y12+receptor+regulates+microglial+activation+by+extracellular+nucleotides.">The P2Y12 receptor regulates microglial activation by extracellular nucleotides.</a> Nat Neurosci. 9 (12), 1512-1519 (2006).</li><li>Gosselin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), (2017).</li><li>Lavin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophage+enhancer+landscapes+are+shaped+by+the+local+microenvironment.">Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.</a> Cell. 159 (6), 1312-1326 (2014).</li><li>Hoeffel, G., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+Tissue-Resident+Macrophages.">Ontogeny of Tissue-Resident Macrophages.</a> Front Immunol. 6, 486 (2015).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci U S A. 113 (12), E1738-E1746 (2016).</li><li>Bohlen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Requirements+for+Microglial+Survival,+Specification,+and+Function+Revealed+by+Defined-Medium+Cultures.">Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.</a> Neuron. 94 (4), 759-773 (2017).</li><li>Herz, J., Filiano, A. J., Smith, A., Yogev, N., Kipnis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+Cells+in+the+Central+Nervous+System.">Myeloid Cells in the Central Nervous System.</a> Immunity. 46 (6), 943-956 (2017).</li></ol>

提交人声明, 这项研究是在没有任何商业或金融关系的情况下进行的, 这可能被理解为潜在的利益冲突。

由于小胶质细胞作为中枢神经系统的前哨免疫单元, 它们对环境变化有高度的响应能力;因此, 在隔离和区域性8期间, 需要非常小心地最小化细胞内的炎症反应。这在这个协议通过速度和温度完成。保持细胞在冰上或在4°c 只要可能大大减少活化, 所以所有离心步骤发生在4摄氏度, 动物正在灌注冰冷灌注缓冲, 和协议已简化, 以减少组织之间的时间细胞的提取和培养。如果评估新隔离细胞的基因表达模式, 则在 trypsinization 前直接从 CD11b immunopanning 皿中收获细胞裂解物, 以尽量减少隔离诱发的变化。应该指出的是, 即使是仔细隔离的小胶质细胞也会在温度升高时立即执行炎症反应, 大概是由于识别与隔离程序内在相关的损伤相关信号。如果协议成功执行, 则预期 2.5-5 x 105单元格/幼鼠。
这里提供的细胞隔离协议是非常具体的隔离 CD11b+ 细胞从灌注的 CNS9。虽然这个细胞的数量主要由小胶质组成, 但它也含有低水平的其他髓细胞群, 如周围巨噬菌、脑膜巨噬细胞、脉络丛巨噬细胞、单核细胞和中性粒细胞10。脉络膜相关的髓细胞在组织离解之前可以通过对脉络丛的识别和去除来消除。脑膜也可以在一定程度上被切除, 但是完全消除脑膜组织在这里所描述的快速隔离的时间范围内是不切实际的。因此, 为了限制不完全切除脑膜的变异性, 我们不去除脑膜。为了最大限度地减少分离材料中的循环单核细胞/中性粒细胞的数量, 有一个干净的灌注也很重要, 我们通常会丢弃不太灌注的组织。即使有出色的灌注, 血液的清除也不是绝对的, 因为在整个纯化过程中, 细胞颗粒中存在少量的红细胞是显而易见的。因此, 小的单核细胞和中性粒细胞被联合纯化, 并在 transcriptomic 的新分离的干细胞的轮廓上作出明显的签名。然而, 循环细胞很快就会死于无血清 TIC 介质中, 并不代表持久性污染物, 尽管它们可以继续存活并在含有文化的血清中增殖。
另一个重要的一点, 以实现一个健康的, 可行的文化是在机械分离步骤照顾。虽然 douncing 杀死许多神经元, 胶质细胞和其他中枢神经干细胞, 小胶质在这种离解中存活得相对不受伤害 (尽管小胶质也可能被过度 douncing 杀死)。如果做得正确, 这个协议就会产生与使用蛋白水解分离获得的细胞收益率, 而在高温下, 组织炎症反应的潜在混杂变量是避免的。通过连续不完整的机械组织离解, 单细胞可以从悬浮中收获, 同时尽量减少机械压力对整体文化的数量。
这里描述的 immunopanning 协议是一种可靠和可重复的方法来隔离小胶质细胞, 但其他可比较的方法是可用的。我们已经取得了类似的结果, 使用磁隔离与磁性活化细胞分类髓鞘损耗和 CD11b 选择。我们这里的重点是 immunopanning 方法, 因为它提供了迄今为止最优质的文化, 需要较少的专业设备, 不需要在细胞培养前立即引入氧化铁-共轭抗体。另一种通常用于胶质隔离的方法是流式细胞术, 它比目前的方法有一个主要优势, 即从其他 CD11b+ 髓系污染物中纯化善意小胶质的表面针对签名标记 (如 TEMEM1198) 的抗体。虽然荧光活化细胞的分类导致极纯细胞的数量, 它也强加给细胞额外的压力, 并可能导致较低的生存能力的文化或降低产量。总的来说, 我们专注于以抗体为基础的方法, 超过无标签密度离心和摇动准备, 以最大限度地重现性和纯度的细胞群体。
本协议针对幼鼠小胶质细胞的培养进行了优化, 可用于培养不同年龄的小胶质细胞;总细胞产量是最大的从 1-2 周大鼠, 在胶质扩张的峰值已经过去了, 并在结构重排之前, 干扰恢复的细胞从旧的组织。小胶质细胞也可以从老鼠和人体组织中分离和培养, 方法是用适当的单克隆抗体替代 CD11b 抗体, 用于小鼠或人的表位。当老鼠和人小胶质细胞在这些培养条件下存活下来时, 它们的形态学差异与大鼠相比。小鼠细胞保留分支形态学, 但有较少精细的过程, 而人类细胞有一个几乎一致的变形形态学时, 隔离和培养根据该程序。
独立的小胶质细胞可以根据所需的化验方法以不同的方式电镀。为获得最佳结果 (如图中所示的活体/死亡化验和形态学电影), 3.5 x 103细胞被镀在24良好阴离子/阳离子涂层组织培养板的中心, 在10分钟的胶原蛋白 IV 涂层后上述议定书所述。或者, 对于需要大量细胞的实验, 如 RNA 隔离, 3-5 x 104细胞可以被镀每一个 24-井板后10分钟到 1 h 胶原蛋白涂层。与斑点治疗的细胞相比, 在较高密度下镀的细胞的生存能力略有降低, 形态学也较不复杂, 可能是由于对隔离的反应而分泌的炎症信号的影响。对于免疫组化或影像学要求玻璃盖玻片, 细胞表现出最佳的生存和形态学时, 盖玻片第一次涂上 PDL, 然后涂在胶原 IV 如上文所述。对于需要大量条件的化验, 细胞的生存能力也很高, 在96井或384井胶原涂层板。在所有情况下, 细胞开始旋转或双极性, 开始获得 3-4 天左右的进程, 并采取 5-10 天来展示在图 1和影片 1中显示的最大分支形态学。
虽然这个协议导致一种文化, 显示动态监视行为和形态学类似于静止的小胶质细胞在体内, 这些单元不能完全保留的专门基因表达签名的小胶质体在体内。在培养细胞中观察到的许多持续性基因变化反映了在胚胎或发炎的 CNS9中常见的改变。因此, 本议定书在了解小胶质细胞在培养和血清诱发变化中生存所需的条件方面迈出了一步, 但需要进一步研究, 以了解中枢神经系统所提供的微调胶质的外部信号。属性和基因表达。

作为中枢神经系统实质的巨噬细胞, 小胶质与大量的神经回路和神经胶质信号网络相互作用。通过突触修剪、凋亡细胞间隙和与神经元过程的瞬态相互作用, 它们在大脑的发育和稳态中发挥重要作用1,2。小胶质细胞是早期神经损伤反应者, 将其长而薄的过程扩展到病变部位, 以协调炎症反应并限制出血3,4。胶质形态和功能的变化在急性和慢性中枢神经系统损伤中普遍存在, 小胶质细胞在多种疾病状态下表现出改变的形态学、定位和炎症介质的表达1。人类遗传研究表明, 改变神经退行性疾病风险的突变, 通常主要或完全由小胶质细胞在完整的中枢神经系统表达, 指出小胶质细胞在疾病发病机制或进展中的关键作用5.鉴于他们在伤病和疾病中的突出地位, 进一步了解胶质生物学是发展新的治疗方法的一个高度优先事项。
对胶质生物学认识的许多重要进展, 都是通过推断其他巨噬细胞的研究中发现的技术和机制, 包括培养方法, 基因表达谱, 以及功能的定义。/形态状态。虽然广义巨噬细胞功能经常在中枢神经系统的景观中以惊人的方式发挥出来, 小胶质是他们自己高度专业化, 展示了分支形态学和独特的基因表达签名, 使他们除了其他组织巨噬细胞6。小胶质细胞具有与大多数其他组织巨噬菌不同的血统;他们殖民中枢神经系统在早期胚胎的原始造血和自我更新的整个生命, 独立的贡献从明确的造血7。成年小胶质细胞的完全成熟基因表达签名直到第二个产后周8才实现。来自周围组织的环境提示在口述组织特异性巨噬细胞特征6中起着重要作用, 在中枢神经系统中, 它包括受血脑屏障9所赋予的血液传播因子的有限接触。
充分理解胶质对中枢神经系统稳态和疾病的贡献的一个障碍是综述在体外发现的成熟小胶质细胞的特殊性质的困难, 即在体内的纯化电池为。许多方法已经发展, 以分离和培养完整的小胶质细胞, 但大多数方法依赖于血清, 以支持生存。我们已经表明, 血清, 这是一个固有的变量试剂含有大量的生物活性分子, 是特别成问题, 当与小胶质细胞, 因为它促进变形形态学, 增加增殖, 并增加的吞噬功能9经常看到体内当小胶质细胞暴露于血脑屏障中断后的血液传播因素。根据这些指标, 血清暴露的细胞在损伤或疾病状态下类似小胶质, 但当小胶质体在含有 CSF-1 (或 IL-34)、TGF β、胆固醇和亚硒酸的定义生长培养基中培养时, 这种改变就会减少。
本议定书提供了有关在无血清条件下培养幼鼠小胶质细胞的细节, 与最近发表的工作9有关。该协议已为产后 21-30 (P21-P30) 的大鼠进行了简化, 但可用于将小胶质细胞从任何年龄的老鼠和老鼠中分离出来, 尽管产量和总体生存能力将因动物的种类和年龄而异。当使用稍未成熟的小胶质细胞 (〜 P9) 时, 最大产量和最佳生存能力得到实现, 其产量和活力逐渐减少到成年动物的某些较低水平。小胶质细胞也可以从老鼠身上分离出来, 但我们发现, 与无血清培养的小鼠细胞相比, 鼠体细胞显示出的分支形态的产量、活力和复杂性显著提高。年龄大于 P50 的动物没有被这个协议评估。此 immunopanning 隔离程序已经过优化, 以最小化在隔离过程中胶质转录剖面的变化, 并最大限度地提高细胞的下游生存能力。使用这些技术和媒体配方, 高活力的初级文化可以持续数周。在这些条件下培养的小胶质细胞具有高度分支的形态学, 包括快速延长和缩回过程, 以及相对较低的增殖速率。我们强调了血清暴露对这些特性的意义, 并讨论了该方法相对于其他方法的优缺点。

<table>
	<tbody>
		<tr>
			<td>Rat: Sprague-Dawley</td>
			<td>Charles River</td>
			<td>Cat# 400</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-rat CD11b monoclonal (clone OX42)</td>
			<td>Bio-Rad</td>
			<td>Cat# MCA275R</td>
			<td>Panning: 1:1,000; Staining: 1:500</td>
		</tr>
		<tr>
			<td>Goat polycolonal anti-Iba1&#160;</td>
			<td>Abcam</td>
			<td>Cat# AB5076</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-Ki67&#160;</td>
			<td>Abcam&#160;</td>
			<td>Cat# AB15580</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-mouse 594</td>
			<td>Invitrogen</td>
			<td>Cat# 11055</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-goat 488</td>
			<td>Invitrogen</td>
			<td>Cat# A-21203</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-Rabbit 647</td>
			<td>Invitrogen</td>
			<td>Cat# A-31573</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Triton-X (detergent in ICC staining)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 28313</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td>Galen Laboratory Supplies</td>
			<td>Cat# GAG-HS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma&#160;</td>
			<td>Cat# M3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from milk solids</td>
			<td>Sigma</td>
			<td>Cat# P6838</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;2</td>
			<td>Peprotech</td>
			<td>Cat# 100-35B</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine IL-34</td>
			<td>R&#38;D Systems</td>
			<td>Cat# 5195-ML/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>Cat# 700000P</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Corning&#160;</td>
			<td>Cat# 354233</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 90260</td>
			<td></td>
		</tr>
		<tr>
			<td>11(Z)Eicosadienoic (Gondoic) Acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 20606</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM dye</td>
			<td>Invitrogen</td>
			<td>Cat# C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer-1</td>
			<td>Invitrogen</td>
			<td>Cat# E1169</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>Cat# DPRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>GE Healthcare</td>
			<td>Cat# 17-5445-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>Cat# 21041-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Gibco</td>
			<td>Cat# 15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>Cat# 25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>Cat# 16634</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>Cat# S-5261</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Gibco</td>
			<td>Cat# 11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>Cat# 0100-20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria Plates&#160;</td>
			<td>VWR</td>
			<td>Cat# 62406-456</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Stock reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 10 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 5 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2.5 mg/mL in H2O 
			Concentration used: 1:25,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:100 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>TGF-b2</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2 mg/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1.5 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Oleic acid/Gondoic acid</td>
			<td></td>
			<td></td>
			<td>Reconstitution: Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 50 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +) 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Douncing Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++ 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Panning Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 2 mg/mL of peptone from milk solids in DPBS++</td>
		</tr>
		<tr>
			<td>Microglia Growth Medium (MGM)</td>
			<td></td>
			<td></td>
			<td>Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite 
			Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr>
			<td>Collagen IV Coating</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing 2 &#956;g/mL collagen IV</td>
		</tr>
		<tr>
			<td>Myelin Seperation Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2 
			Comments: Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr>
			<td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid 
			Comments: Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.</td>
		</tr>
	</tbody>
</table>

isolation and culture of rodent microglia to promote a dynamic ramified morphology in serum-free medium

小胶质细胞代表了 5-10% 的所有中枢神经系统 (CNS) 单元, 并越来越引起关注, 因为他们的贡献在发展, 稳态和疾病。尽管巨噬细胞已经被详细研究了数十年, 但小胶质细胞, 中枢神经系统的组织驻留巨噬菌的特殊特征仍然很大程度上是神秘的, 部分原因是对成熟胶质的重述能力的限制。文化中的属性。在这里, 我们说明了一个简单的程序, 快速分离的纯小胶质细胞从成熟的啮齿动物大脑。我们还描述了无血清的文化条件, 支持高水平的胶质生存时间。在这些定义中等条件下培养的小胶质细胞具有精心的分支过程和动态监测行为。我们说明了血清暴露对小胶质细胞的一些影响, 并讨论了这些无血清的文化与血清暴露的文化和小胶质酶在体内的比较。

本协议描述了一种从幼年大鼠中培养高纯度分支小胶质细胞的方法。类似的结果可以得到使用不成熟, 围产期和成年动物, 如下面所述。由于细胞隔离通常包括微妙的细微差别和许多细胞死亡的机会, 我们使用质量控制检查站来帮助确定在各个步骤的成功。我们通常在隔离协议 (图 1A) 的多个步骤中使用 hemocytometer 监视单元格悬浮。成功的隔离应该产生 2.5-5 x 105细胞/幼兽。从 P7-P15 的动物显示出产量接近 5 x 105细胞/动物, 而从比 P30 大的动物中分离出来的细胞产量接近 2.5 x 105细胞/动物。除了在整个协议中监测总的细胞计数外, 重要的是要确定孤立细胞的整体健康, 这是很困难的, 因为在这种方式下分离的小胶质在培养的头几天内有一个圆形或双极形态学。.在这里, 我们包括了 P25 小胶质细胞在分离后的第一6天内, 在 tic 和 tic 中的相图, 其中含有10% 胎小牛血清 (FCS) (图 1B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57122/57122fig1.jpg"> 图 1: 隔离协议期间的细胞健康评估和超过6天的文化.(A) 相对比图像, 在单元格隔离过程的指定步骤中演示质量控制检查点。细胞悬浮的图像通过混合9µL 的细胞悬架与1µL 0.4% 台盼蓝前加载到 hemocytometer。其他图像显示平移盘本身, 所有图像显示为一个字段在20X 放大倍数。鳞片杆, 200 µm 米高梅, 小胶质生长培养基。(B) 无血清 TIC 生长培养基或 tic 加10% 胎小牛血清 (FCS) 的胶质形态学改变和增殖超过6天的时间过程。CD11b-immunopanned 细胞被镀在胶原膜的阴离子/阳离子涂层组织培养板上 (参见材料目录), 同一字段的图像的相位对比每24小时, 100 µm. TIC, TGF-β2/IL-34/含有生长培养基的胆固醇。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57122/57122fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
常规评估分离细胞的健康和生存能力对重现性和实验的成功具有重要意义。在这里, 我们展示了小胶质细胞从一个 P21 动物培养在米高梅, tic, 或 tic 补充与 10% FCS 后7天体外(DIV) 与可行性试验。如果隔离成功, 在 7 DIV 之后, 在 TIC 中培养出 80-90% 的生存能力. 细胞在血清中的存在最终攀登到接近100% 的生存能力由这项措施, 然而这是人为地夸大由迅速增殖血清暴露的细胞和这些文化中死细胞的快速吞噬功能 (图 2A)。除了强健的生存, TIC 培养细胞也显示了分支形态学时, 与 FCS 处理细胞 (图 2B, C)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57122/57122fig2.jpg"> 图 2: 美高梅、tic 或 tic + FCS 中小胶质细胞在文化中的存活时间超过七天.(A) 细胞与 P21 大鼠分离, 培养在小胶质生长培养基 (米高梅), TGF-β2/IL-34/Cholesterol 含生长培养基 (tic), 或 TIC + 10% 胎小牛血清 (FCS)。在每个时间点, 培养基辅以 calcein AM (1.33 µM 最终) 和溴 homodimer (2.5 µM 最后) 15 分钟, 分别标记活细胞和死细胞。使用自动 ImageJ 脚本在每个字段中计算单元格。(B和C) 在 7 DIV 之后, 没有媒体更改, 在 tic (B) 或 tic 中生长的单元格 (C) 被染色, 以标记活和死细胞。使用 10X (左) 或 40X (右) 目标拍摄了代表性图像。缩放栏, 40 µm. 误差线表示平均值 (SEM) 的标准误差。此数字是从忆读过et. 修改的。9并与权限一起使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57122/57122fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了证明高纯小胶质细胞的成功分离和培养, 我们从 P21 大鼠中分离出小胶质体, 并在 tic 或 tic 培养这些干细胞, 补充 10% FCS 8 天。然后用胶质标记 (Iba-1 和 CD11b) 和增殖标记 (Ki67图 3A) 对细胞进行固定和染色。除了染色的细胞标记, rna 序列的新鲜孤立的细胞可以提供一个强大和敏感的工具, 以评估的 RNA 剖面的细胞, 并可以帮助通知的起始纯度的文化。除了 CD11b+小胶质细胞 (图 3B) 外, CD11b immunopanning 通常会导致 CD11b+中性粒/单核细胞的少量百分比。这些细胞在几天后会死于 TIC, 但会使早期时间点的分析复杂化。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57122/57122fig3.jpg"> 图 3: 免疫组化和 RNA-下向培养细胞的纯度(A) 小胶质细胞从 P21 大鼠中分离出来, 被镀到 PDL/胶原膜玻璃盖玻片, 并在 TGF-β2/IL-34/Cholesterol 中培养, 生长培养基 (TIC) 8 天。细胞在室温下固定与4% 多聚甲醛 (粉煤灰) 10 分钟, 免疫染色的 Iba-1 (1:500), CD11b (1:1500), Ki67 (1:500)。简单地, 固定的细胞被阻拦了以0.1% 洗涤剂 (参见方法表为细节) 和10% 正常驴子血清 (在室温下1小时)。然后用0.1% 洗涤剂中的主要抗体孵化出细胞, 在4摄氏度一夜之间1%。细胞在 PBS 中每5分钟洗三次。然后, 在室温下, 细胞在0.1% 洗涤剂中以1:500 的二级抗体进行孵化, 其中1% 为1小时。细胞在 PBS 再次被洗涤了三次并且安装了以 DAPI 包含的安装的媒介。具有代表性的图像以40X 倍的放大率拍摄。缩放条, 50 µm. (B) 用新鲜隔离和培养的细胞表达典型细胞类型的特定转录。小胶质细胞从 P21 大鼠和裂解直接从 immunopanning 盘中分离出来, 用于 rna 的分离, 其他主要的中枢神经干细胞类型 (星形胶质细胞、神经元、少突胶质和内皮细胞) 的 rna 的标记也显示出最小的污染CD11b-immunopanned 细胞。中性粒细胞标记被发现在新鲜隔离的电池 (黑色), 但在无血清培养 (灰色) 丢失。培养细胞表达高水平的常见巨噬细胞标记, 但定义的专门胶质签名的标记是快速 downregulated 的培养细胞。误差条表示平均值 (SEM) 的标准误差。数据从忆读过et. 修改。9并与权限一起使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57122/57122fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
染色为生存能力和细胞标记允许确定细胞的生存和纯净, 但提供有限的信息关于文化的动态。在这里, 我们提供了一个 P21 鼠小胶质细胞在 TIC 14 DIV 后的时间推移电影。两周后, 无血清的文化显示动态监视行为, 快速的过程扩展和撤消在整个盘子 (参见影片 1)。此外, 我们以前发现, 血清暴露改变这些静止状态的小胶质细胞, 导致持久的形态学和吞噬性的变化。我们在 TIC 中包括了一部培养了5天的小胶质细胞的电影, 然后暴露在 10% FCS (参见影片 2)。
<img alt="Movie 1" src="/files/ftp_upload/57122/57122mov1.jpg"> 影片 1: 定义中的小胶质细胞区域性显示具有动态进程的分支形态学。小胶质细胞从 P21 大鼠中分离出来, 斑点镀在胶原膜上的阴离子/阳离子涂层组织培养板上, 在 TIC 培养14天。在 14 DIV, 每3分钟收集图像。影片在6帧/秒播放, 右下角的时间戳表示图像在 h 中的持续时间: min. 影片是从忆读过et. 复制的。9并与权限一起使用。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57122/Movie_1.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Movie 2" src="/files/ftp_upload/57122/57122mov2.jpg"> 影片 2: 血清暴露会触发定义中的胶质文化中的快速形态学变化。小胶质细胞从 P21 大鼠中分离出来, 斑点镀在胶原膜上的阴离子/阳离子涂层组织培养板上, 在 TIC 培养5天。在 5 DIV, 每5分钟收集图像基线运动/形态学记录, 然后在 2:55 (2 小时, 55 分钟), 一个50% 的媒体变化, 以增加 FCS 到最后浓度10%。细胞被成像了额外的7小时。影片在6帧/秒播放, 右下角的时间戳表示图像在 h: min 的持续时间.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57122/Movie_2.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>

Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

由于缺乏对成熟的体内小胶质细胞的特性进行重述的胶质文化模型, 胶质的详细理解的努力受到了阻碍。该协议描述了一种隔离和培养方法, 旨在维持高分支成熟大鼠小胶质细胞在确定培养基条件下的健壮生存。

鼠小胶质细胞的分离培养促进无血清培养基动态分支形态学的研究

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

isolation-culture-rodent-microglia-to-promote-dynamic-ramified

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.8K 次观看.  Stanford University. 由于缺乏对成熟的体内小胶质细胞的特性进行重述的胶质文化模型, 胶质的详细理解的努力受到了阻碍。该协议描述了一种隔离和培养方法, 旨在维持高分支成熟大鼠小胶质细胞在确定培养基条件下的健壮生存。

视频: Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

动态定量感觉测试表征中央疼痛处理

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

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 ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...