这项研究得到了国家多发性硬化协会 (RG4591A1/2) 和国立卫生研究院 (R03NS06740402) 的赠款的支持。作者感谢 Ivan 博士及其实验室成员提供实验室空间、设备和建议。

本研究中使用的小鼠根据纽约州立大学南部医学中心实验室动物资源 (DLAR) 协议15-10492 号的指导方针予以照顾。
注: 原少突胶质与新生儿 (P5-P7 野生型 C57Bl/6N) 小鼠分离。在这一阶段, 不成熟的生命线构成了80% 以上的啮齿动物白质, 确保高细胞产量。所有缓冲和试剂组合物都可在材料表的末尾提供。
1. 盖玻片准备
注: 聚 d-赖氨酸 (PDL)/层粘连蛋白涂层盖玻片应在 OL 隔离之前准备好。
<ol><li>将 #1 德国玻璃盖玻片放入50毫升锥形管中, 并加入35毫升70% 乙醇以清洁它们。</li>
	<li>关闭管, 把它放在一个 nutating 搅拌机, 并在室温下孵化至少30分钟, 同时轻轻摇摆。盖玻片可以在一夜之间孵化。</li>
	<li>丢弃70% 乙醇。</li>
	<li>用去离子水清洗盖玻片3次, 每次用真空吸水的时候都要去除水分。</li>
	<li>增加30-35 毫升的 PDL 50 µg/毫升的盖玻片在50毫升管, 足以覆盖盖玻片。</li>
	<li>将含有盖玻片的50毫升管放在 nutating 搅拌机上, 在室温下孵育至少30分钟, 同时轻轻摇动。</li>
	<li>用去离子水清洗盖玻片3次, 每次用真空吸水时, 都要去除水分。</li>
	<li>将盖玻片转移到含有去离子水的60毫米培养皿中。</li>
	<li>使用 P200 吸管尖将盖玻片排列成覆盖培养皿整个表面的层。</li>
	<li>小心, 从盘子里取出水, 抽吸真空。</li>
	<li>除去盖玻片周围的多余水分, 让他们一夜之间用盖子打开, 在罩下紫外线照射下晾干。 注: 在无菌培养皿中, PDL 涂布玻璃盖玻片可储存在4摄氏度, 长达三月。</li>
	<li>将盖玻片放入24井板中, 每井一条, 使用细钳。</li>
	<li>将盖玻片移动到井的中心, 确保边缘不触及井壁。</li>
	<li>在 B27NBMA 中添加100µL 10 µg/毫升层粘连蛋白 (参见材料表), 从每个盖玻片的中心开始, 以圆形的方式向边缘移动以覆盖所有区域。 注: 由于层粘连蛋白参与了 OL 维护, 并促进分化为成熟的生命线, 它是体外区域性41,42,43,44的重要的 OL 生存因素。</li>
	<li>在 5% CO2的37°c 孵化器中孵化出至少1小时的板材, 或者直到生命线准备好进行电镀。</li>
</ol>2. 鼠大脑皮层离解
<ol><li>牺牲产后5-7 天老 C57Bl/6N 鼠幼崽通过快速斩首与剪刀以前清洗过70% 乙醇。 注: 5-6 只新生小鼠脑皮质用于每种制剂。平均来说, 一只老鼠的大脑产生 1-1. 5 x 106生命线。</li>
	<li>用小的解剖剪刀剪下中线的头皮皮肤, 再缩回以暴露头骨。</li>
	<li>小心地沿着中线切开头骨, 从头骨后部的开口开始朝向前额区域, 用剪刀抬起, 以避免损伤大脑。然后, 用头骨后部的开口沿着头骨的底部切开每个眼窝。</li>
	<li>使用细钳轻轻地戏弄皮层远离中脑, 并转移到一个60毫米的组织培养皿含有7毫升的 B27NBMA。对每个大脑重复步骤2.2 到 2.4, 将解剖过的皮层集中。</li>
	<li>将皮质转移到一个新的60毫米组织培养皿中, 内含5毫升离解缓冲液 (B27NBMA、20-30 u/毫升木瓜蛋白酶和 2500 U DNase I)。</li>
	<li>将皮质切成小块约1毫米3使用 #15 手术刀刀片和孵化20分钟在37°c, 5% CO2孵化器。</li>
	<li>添加1毫升牛生长血清 (BGS), 以停止酶反应。</li>
	<li>使用10毫升血清吸管将皮质与离解介质转移至15毫升圆锥管。</li>
	<li>轻轻地开始用10毫升的血清吸管吹打6-8 次, 慢慢地将脑组织分离出来, 用小心最小化气泡。</li>
	<li>允许组织块安定2-3 分钟和转移上清到新鲜的管子。</li>
	<li>添加3毫升的 B27NBMA 含有 10% BGS/2500 U DNase I 到组织颗粒。</li>
	<li>使用5毫升血清吸管轻轻地分离脑组织, 而吹打上下6-8 次。注意尽量减少气泡。</li>
	<li>允许组织块解决2-3 分钟。</li>
	<li>将上清液转移到新鲜的管子上。添加3毫升的 B27NBMA 含有 10% BGS/DNase I 的组织颗粒。</li>
	<li>用 P1000 吸管尖轻轻地分离脑组织, 同时吹打媒体和脑组织的混合。重复步骤2.13 和 2.14, 直到没有大块的组织保持或直到 B27NBMA 包含 10% BGS/DNase 我累了, 谁先来。注意尽量减少气泡。</li>
	<li>池细胞悬浮与早先上清液。</li>
	<li>将 70-µm 细胞过滤器放置在50毫升管中。使用10毫升血清吸管, 通过在70µm 细胞过滤器轻轻地通过池细胞悬浮。</li>
	<li>通过添加1毫升含有 10% BGS/DNase i 的 B27NBMA 来清洗细胞过滤器。</li>
	<li>丢弃细胞过滤器。用含有 10% BGS/DNase i 的 B27NBMA, 将体积带到30毫升。</li>
	<li>将细胞悬浮液转移到两个15毫升锥形管上。离心机细胞悬浮10分钟在 200 x g。</li>
	<li>移除上清液, 不让暴露在空气中的细胞离开。由于细胞碎片, 上清将多云。添加3毫升的 B27NBMA 含有 10% BGS 的颗粒。</li>
	<li>用 P1000 吸管仔细分离细胞颗粒。将体积带到15毫升, 含 B27NBMA 10% BGS。</li>
	<li>通过细胞悬浮在一个新鲜的40µm 细胞过滤器。</li>
	<li>通过添加含有 10% BGS 的 B27NBMA 1mL 来清洗细胞过滤器。丢弃细胞过滤器。</li>
	<li>将体积带到30毫升, 含 B27NBMA 10% BGS。</li>
	<li>将细胞悬浮液转移到 2 x 15 毫升锥形管上。离心机细胞悬浮10分钟在 200 x g。</li>
	<li>除去大部分上清液, 而不让暴露在空气中的细胞离开。并用重悬5毫升的冰冷磁细胞分选 (MCS) 缓冲细胞颗粒。</li>
</ol>3. 细胞计数和生存能力的测定
<ol><li>稀释100µL 的细胞悬浮与400µL 的台盼蓝溶液在1.5 毫升离心管, 以达到1:5 稀释 0.4% (瓦特/v)。</li>
	<li>将盖玻璃放在 hemocytometer 室上, 用 P10 吸管和避免溢出填充两个腔, 用10µL 细胞稀释。溶液将通过毛细管作用在盖玻璃下通过。</li>
	<li>将 hemocytometer 放在显微镜舞台上, 用40X 放大器调整焦距。 注意: 细胞计数是使用手持计数器在每五个正方形 (四个角落和一个中心) 记录。只有非可行的细胞吸收染料, 并出现蓝色, 而活和健康的细胞出现圆形和屈光, 不吸收蓝色染料, 允许确定的数量的可行和总细胞每毫升。</li>
</ol>4. 隔离 O4+少突胶质
<ol><li>离心机细胞悬浮在 200 x g 为10分钟。</li>
	<li>用真空抽吸法小心丢弃上清液, 避免细胞暴露于空气中。</li>
	<li>并用重悬每 1 x10 7 总单元格中的90µL 的 MCS 缓冲区中的颗粒, 然后添加10µL 的 anti-O4 珠, 每 1 x10 7 单元格。</li>
	<li>用手指轻轻地弹15毫升圆锥管, 将细胞悬浮和珠子混合4-5 次。</li>
	<li>在4摄氏度孵育15分钟的混合, 用手指 4-5 timesevery 5 分钟弹15毫升圆锥管。</li>
	<li>将每 1 x 107单元格中的 MCS 缓冲区的2毫升缓冲液轻轻地添加到管中来清洗混合。离心机的混合在 200 x g 10 分钟。</li>
	<li>小心使用真空吸尘, 避免细胞暴露在空气中, 从而丢弃上清液。并用重悬每个 1 x10 7 单元格中的500µL 的 MCS 缓冲区中的细胞颗粒。</li>
	<li>将磁选机连接到磁选机支架上。将选择列附加到磁选器上, 并将40µm 单元格滤网放在该列的顶部。将两个15毫升或一个50毫升圆锥管置于分离柱下面, 以收集流过的水流。</li>
	<li>用3毫升的 MCS 缓冲器预冲洗40µm 细胞过滤器和分离柱, 让缓冲器在柱子上运行, 不让它干。</li>
	<li>添加细胞悬浮和珠子的混合到细胞过滤器, 并进入选择栏。</li>
	<li>用1毫升的 MCS 缓冲器清洗40µm 细胞过滤器。</li>
	<li>丢弃细胞过滤器, 让细胞、珠子和缓冲器混合在一起, 不让它干。</li>
	<li>用3毫升的 MCS 缓冲器和1次用 OL 扩散介质清洗分离柱3次。</li>
	<li>将分离柱从磁选机中取出, 快速将其放入15毫升锥形管中, 立即加入5毫升的 OL 扩散介质。</li>
	<li>将柱塞放在立柱顶部, 并将被标记的细胞冲出15毫升锥形管。</li>
	<li>删除100µL 细胞悬浮, 以确定细胞计数和可行性使用台盼蓝和 hemocytometer 如第3节所示。 注意: 80% 以上的细胞活力被认为是可以接受的, 可以继续使用苏丹生命线的文化, 但生存能力大于90% 是最佳的。</li>
</ol>5. 隔离 O4+少突胶质的电镀
<ol><li>使用 OL 扩散介质, 将细胞悬浮液稀释至欲望电镀密度 (每盖玻片 5 x10 5 单元格)。 注意: OL 播种密度非常重要, 因此在进行前应使用组织培养显微镜确定合适的细胞密度。播种密度低于1万细胞/盖玻片可能导致细胞存活不良。在镀层密度为1万到2.5万时, OL 形态分化缓慢45。我们在至少5万细胞/盖玻片的播种密度下, 以确保细胞存活。</li>
	<li>将含有层粘连蛋白的盖玻片24井板移动到组织培养罩中。</li>
	<li>从每个盖玻片中去除层粘连蛋白, 并用100µL 的 OL 悬浮液取代。</li>
	<li>在37°c/5% CO2孵化器孵化的生命线, 最大45分钟, 以促进对盖玻片的 OL 黏附力。</li>
	<li>孵化后, 从孵化器中取出24井板。将24井板块的每一个井与500µL 的 OL 扩散介质一起淹没。</li>
	<li>将生命线恢复到37°c/5% CO2孵化器, 以增加24小时。</li>
	<li>24小时后, 去除扩散介质, 用 OL 分化培养基替代, 诱导分化。</li>
	<li>把生命线放回孵化器中, 不要移除, 直到细胞准备好固定。 注意: pH 值的变化对苏丹生命线行动和降低细胞活力是有害的, 因此应该避免从孵化器中不必要地去除生命线的文化。</li>
</ol>6. 免疫荧光染色
<ol><li>为检测细胞表面标记物 (NG2、O1 和 O4), 从油井中取出介质。</li>
	<li>添加250µL 的小鼠杂交瘤上清液含有主要抗体对 O1 (未稀释) 13 和 O4 (未稀释) 13 和兔多克隆对 NG2 (1:150 稀释在 B27NBMA)。 注: 如果使用商用 anti-O1 和 anti-O4 抗体, 最好利用制造商建议的稀释。</li>
	<li>在37°c/5% CO2孵化器中, 用主抗体在45分钟内孵化细胞。</li>
	<li>用 0.05% Tween-20/1X PBS 清洗两次。在室温下用4% 多聚甲醛 (4% 粉煤灰) 固定细胞, 10 分钟。</li>
	<li>每5分钟洗三次, 0.05% Tween-20/1X PBS。</li>
	<li>稀释 Alexa 488-共轭山羊抗鼠 IgM 或抗兔 IgG 二级抗体 (1:400) 在 B27NBMA。</li>
	<li>从盖玻片中卸下 0.05% Tween-20/1X PBS。在黑暗的室温下, 用二级抗体在室温下孵化细胞1小时。</li>
	<li>每5分钟洗三次, 0.05% Tween-20/1X PBS。</li>
	<li>用于检测内部标记 (GFAP) 的双色染色, 在室温下取出 0.05% Tween-20/1X PBS 和块/通透缓冲器15分钟的块状/permeabilize 细胞。否则继续执行步骤6.17。</li>
	<li>删除块/通透缓冲区, 并添加250µL 鸡抗胶质 (1:100) 稀释在块/通透缓冲。</li>
	<li>室温下以1小时的抗胶质细胞孵化。每5分钟洗三次, 0.05% Tween-20/1X PBS。</li>
	<li>稀释 Alexa 594-共轭山羊抗鸡 IgG 二级抗体 (1:400) 在 B27NBMA。</li>
	<li>从盖玻片中卸下 0.05% Tween-20/1X PBS。在黑暗的室温下, 用二级抗体在室温下孵化细胞1小时。</li>
	<li>每5分钟洗三次, 0.05% Tween-20/1X PBS。</li>
	<li>Counterstain 与 4 ', 6-diamidino 2-苯基吲哚 (DAPI), 并装入包含 antifade 的 DAPI 安装介质的细胞。</li>
	<li>让盖玻片在黑暗中过夜, 在室温下治疗。</li>
	<li>用荧光显微镜对染色的细胞进行图像处理。 注意: 在本协议中, 使用均匀曝光时间对单元格进行40X 放大成像。</li>
</ol>

作者没有披露。

本文提出了一种高效分离高纯度未成熟小鼠少突胶质培养的方法。与以前发布的协议39、40相比, 此方法产生了更高的纯度, 具有低得多的 GFAP 阳性星形胶质细胞和其他非特征细胞的极低百分比。重要的是要指出, 这些是未成熟的生命线行动已经致力于 oligodendroglial 血统。因此, 这些细胞对于研究分化的早期阶段是不有用的。
Dincman 协议40的一项修改是从我们的扩散介质中去除碱性成纤维细胞生长因子 (bFGF)。据报道,体外, bFGF 作为一个强大的有丝分裂因子, 不仅抑制 OPCs 的分化, 但也减少进程的数量46,47。此外, bFGF 增加了 oligodendroglial 谱系48传代细胞的少突胶质分化和增殖。因此, 在 Dincman 研究40中观察到的 O4+单元数的减少和 A2B5+和 NG2+单元格数目的增加是合理的, 这可能是由于 bFGF 在培养中的作用。
尽管使用 O4 绑定作为隔离单元格的方法, 但只有59% 的单元格在 1DIV O4+ 。这些结果在质量上类似于 Dincman 和同事40 , 他们发现使用 O4 绑定进行隔离时, 在 1DIV O4+的单元格略少于50%。结果表明, 血小板生长因子延迟有丝分裂后的发展通过瞬态恢复 O4+GalC 祖 A2B5 +O4-前祖像细胞, 随后分化, 即使在继续存在的血小板形成49. 因此, 很可能在第一天的扩散过程中, 在体外中, 一些未成熟的苏丹生命线行动可能会经历从 O4+NG2+到 O4 NG2+的逆转, 同时仍保留承诺少突胶质血统。此假说由 Dincman 的发现40支持, 即在隔离大约80% 的单元格 O4+后立即进行。
在 3DIV, 此协议产生比其他39、40所描述的更高的 OL 纯度, 尽管我们没有对它们的方法进行头部与头部的比较。与 Dincman et . 的协议比较40, 我们的文化中的 GFAP 阳性细胞数量越少, 可能是由于扩散媒介中没有生长蛋白的缘故。金刚砂和 Dugas 协议39依赖于细胞通过培养皿的连续通道;星形胶质细胞可以很容易地连接到培养皿50 , 并可以在每个连续通道的细胞在 immunopanning 协议期间进行。所提出的方法不冒险引入污染的星形胶质细胞在我们的纯化分数。
我们的改进方法比其他两个要快4小时。我们通过消除其他协议所包含的不必要步骤和试剂来实现这一目标。例如, 与 Dincman 协议40(需要5-6 小时) 相比, 我们省略了用于神经组织离解的工具包, 以及用大鼠抗鼠 IgM 珠子对 OL 细胞悬浮的孵化, 以去除死细胞。相反, 我们进行了小鼠大脑皮层与木瓜蛋白酶的分离, 死细胞通过执行低速 centrifugations (200 x g) 去除。
从金刚砂协议39的一个主要修改, 大约需要9小时, 是删除 immunopanning 选择和取消对直接 O2/CO2线的要求, 用于在组织中预平衡木瓜蛋白酶缓冲消化。相反, 我们在 CO2孵化器中使用了磁选择和直接孵化小鼠大脑皮层和木瓜蛋白酶缓冲的组合。
虽然没有直接测试在本研究中, anti-O4 微球也可以被用来分离从大鼠, 人类和转基因小鼠脑组织的生命线。这突出了技术的通用性, 使用多个组织来源的生命线行动隔离, 并有可能造福研究实验室感兴趣的工作, 从除鼠标以外的来源。
总之, 本研究中所描述的改进的 OPC 隔离方法提供了一种快速而具体的选择, 以获得可用于髓鞘形成和脱髓鞘疾病研究的原发性小鼠少突胶质。

少突胶质 (生命线) 是中枢神经系统 (CNS)1 的 myelinating 细胞。在严格调节的环境中, 主少突胶质的分离和培养是体外研究 oligodendroglia 的重要工具, 也是脱髓鞘疾病 (如多发性硬化症) 的生物学基础2.这需要一个高效且健壮的少突胶质隔离和区域性方法3。在本研究中, 我们利用了一个独特的少突胶质细胞表面标记的表达, 以实现一个快速和具体的改进的隔离技术。
已经确定了四个不同的少突胶质成熟阶段, 每一个发育阶段都表现出独特的细胞表面标记(图 1)。这些单元格表面标记可以通过特定的抗体4、5识别, 并可用于在特定阶段隔离生命线。在第一阶段, 少突胶质前体细胞 (OPCs) 有能力增殖, 迁移, 并明确表达血小板衍生生长因子受体 (血小板 Rα)6, 苷 A2B5, 蛋白多糖NG27, 8, 聚唾液酸-神经细胞黏附分子9和脂肪酸结合蛋白 7 (FABP7) 10. OPCs 具有双极性形态学, 细胞体的对立两极产生的短过程很少, 这是神经前体细胞11的特征。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57543/57543fig1v2.jpg"> 图 1: 在鼠标少突胶质开发过程中单元格表面标记的表达式。A2B5、GalC (O1)、NG2、O4 和血小板因子 Rα等生命线细胞表面标记物可用于在特定发育阶段用特定抗体分离少突胶质。  <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57543/57543fig1v2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
在第二阶段, OPCs 产生 preoligodendrocytes 和表达的细胞膜不仅 OPC 标记, 而且 sulfatide (硫酸 galactolipid) 承认 O4 抗体12,13, GPR17 蛋白14,一直持续到未成熟的少突胶质 (OL) 阶段。在这个阶段, preoligodendrocytes 扩展多极短过程。Preoligodendrocytes 是大鼠和鼠脑白质中 2 (P2) 的主要 OL 阶段, 其小种群为未成熟的生命线15。
在第三阶段, 未成熟的生命线继续表达 O4, 失去 A2B5 和 NG2 标记的表达, 并开始表达 galactocerebroside C16。在这个阶段, 苏丹生命线行动致力于 oligodendroglial 谱系, 成为后有丝分裂细胞与长分支分支17,18。未成熟的 OL 构成80% 以上的啮齿动物白色物质在 P7 和此时第一个 MBP+单元格被观察15,19,20,21。因此, 在 P7 中分离生命线能保证细胞高产。
在 OL 发育的最后和第四阶段, 成熟的 myelinating 蛋白 (髓鞘碱性蛋白 (MBP), proteolipid 蛋白 (), 髓鞘相关糖蛋白 (MAG) 和髓鞘少突胶质糖蛋白 (MOG) 22, 23 ,24,25,26。在这个阶段, 成熟的生命线延伸膜, 形成紧凑的包裹鞘围绕轴突和能够 myelinate。这与观察老鼠和老鼠大脑的情况一致, 在 P1419,20,21中, MBP+细胞变得越来越丰富。
自从 Fewster 和同事在1967年首次隔离少突胶质27 以来, 已实施了几种方法, 用于从啮齿动物中枢神经系统中分离生命线, 其中包括immunopanning28、29、30、荧光活化细胞分选 (资产管制署) 利用细胞表面特异抗原28,31, 差分梯度离心32,33,34,35和基于不同中枢神经胶质细胞差异粘附的震动方法36,37。然而, 现有的文化方法有局限性, 特别是在纯度、产量和执行程序38所需的时间方面。因此, 需要更有效的少突胶质隔离方法。
本文提出了一种简便、有效的选择方法, 用于磁三 O4+ preoligodendrocytes 细胞与新生小鼠幼崽的分离。此方法是对金刚砂et所报告的技术的修改。39和 Dincman et 。40 , 并提供在大约4小时以上80% 的少突胶质制备纯度。

<table>
	<tbody>
		<tr>
			<td>10ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml syringe Luer-Loc tip</td>
			<td>BD, Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml conical tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22368547</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10H</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm tissue culture plates</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21042</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11042</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-O4 beads- Anti-O4MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-543</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-Transferrin human</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Growth Serum (BGS)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP-1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Peprotech</td>
			<td>450-50</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Desoxyribonuclease I (DNAse I)</td>
			<td>Worthington</td>
			<td>LS002007</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope with an Olympus DP70 camera</td>
			<td>Olympus</td>
			<td>Bx51</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather disposable scalpels</td>
			<td>Andwin Scientific</td>
			<td>EF7281C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>German glass coverslips, #1 thickness, 12mm diameter round</td>
			<td>NeuVitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Aves</td>
			<td>GFAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Invitrogen</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator stand - MACS multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator-MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex PES 0.22&#181;m filter unit</td>
			<td>Millipore</td>
			<td>SLG033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media- Prolong Gold with DAPI</td>
			<td>Thermo Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-cysteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural mouse laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium A</td>
			<td>Invitrogen</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotrophin-3 (NT-3)</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2 antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>D5652</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection column-LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace elements B</td>
			<td>Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine (T3)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>B27NBMA</td>
			<td></td>
			<td></td>
			<td>487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 &#176;C until use.</td>
		</tr>
		<tr>
			<td>B27NBMA + 10% BGS</td>
			<td></td>
			<td></td>
			<td>27 mL B27NBMA; 3 mL Bovine growth serum</td>
		</tr>
		<tr>
			<td>CNTF solution stock (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer&#8217;s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 &#176;C. 
			Working solution (10 &#181;g/ml, 1000X) 
			1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 10&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>d-Biotin stock solution (50 &#181;g/ml; 5000X)</td>
			<td></td>
			<td></td>
			<td>Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 &#181;g/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37&#176;C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10&#181;g/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DNase I stock solution</td>
			<td></td>
			<td></td>
			<td>1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice. 
			2. Filter sterilize on ice 
			3. Aliquot at 200 &#181;l and freeze overnight at -20&#176;C. 
			4. Store aliquots at -20 to -30&#176;C.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (w/o Ca2+ and Mg2+)</td>
			<td></td>
			<td></td>
			<td>Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Forskolin stock solution (4.2 mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 &#181;l) and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salts (HBSS) (Sigma</td>
			<td></td>
			<td></td>
			<td>1. Measure 900 ml of water (temperature 15-20 &#176;C) in a cylinder and stir gently. 
			2. Add the power and stir until dissolved. 
			3. Rinse original package with a small amount of water to remove all traces of the powder. 
			4. Add to the solution in step 2. 
			5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume. 
			6. Keep stirring until dissolved. 
			7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH. 
			8. Add additional water to bring the final volume to 1L. 
			9. Sterilize by filtration using a membrane with a porosity of 0.22 microns. 
			10. Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Insulin stock solution (4000 &#181;g/ml)</td>
			<td></td>
			<td></td>
			<td>Thaw the bottle and aliquot 25 &#181;l per microcentrifuge tube and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Laminin solution</td>
			<td></td>
			<td></td>
			<td>Slowly thaw laminin in the cold (2&#176;C to 8&#176;C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5&#176; C to -20&#176; C in aliquots (e.g. 20 &#181;l) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.</td>
		</tr>
		<tr>
			<td>Magnetic Cell Sorting (MCS) Buffer</td>
			<td></td>
			<td></td>
			<td>Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5&#181;g/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 &#181;m Millex filter. Store the buffer at 4&#176;C until use</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 &#181;l). Store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>NT3 stock solution (1 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1 &#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>PDGF stock solution (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>Poly-D-lysine (1mg/ml; 100X)</td>
			<td></td>
			<td></td>
			<td>Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100&#181;l/tube). Store at -20&#176;C. Prior to use, dilute the 100X stock (1mg/ml) to 50 &#181;g/ml in sterile water.</td>
		</tr>
		<tr>
			<td>Oligodendrocyte proliferation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Oligodendrocyte differentiation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Sato supplement (100X)</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. &#38; Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. &#38; Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and specific immunomagnetic isolation of mouse primary oligodendrocytes

高效、健壮的分离和培养原少突胶质 (生命线)是体外研究 oligodendroglia 的重要工具, 也是脱髓鞘疾病 (如多发性硬化) 的生物学和Pelizaeus Merzbacher 样疾病 (PMLD)。在这里, 我们提出了一个简单而有效的选择方法, 磁隔离三期 O4+ preoligodendrocytes 细胞从新生小鼠幼崽。由于未成熟的 OL 在产后 7 (P7) 的啮齿动物脑白质中占80% 以上, 这种隔离方法不仅保证了高细胞的产量, 而且还对已经致力于 oligodendroglial 谱系的生命线的具体隔离, 减少了可能隔离污染的细胞, 如星形胶质细胞和其他电池从老鼠的大脑。这种方法是对以前报告的技术的修改, 并提供少突胶质的纯度在80% 以上约4小时。

本研究的目的是为 O4+主鼠标少突胶质建立一个改进的隔离方法, 要求对目标单元格进行尽可能少的操作。整个过程从幼崽的安乐死到电镀细胞在盖玻片需要大约 4 h, 并且这里显示的数据代表三个独立实验。组织离解后, 每项独立实验的平均 4.3 0.46 x 107细胞被隔离, 其生存能力为 91% @ 5.6%。在使用 anti-O4 标记磁性微珠磁隔离之后, 平均获得了6.9 个 0.38 x 106生命线 (1-1. 5 x 106每鼠标), 这是 16.2% @ 1.6% 的总细胞最初离解;生存能力为 96.3%, 3.5%。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57543/57543fig2.jpg"> 图 2: Immunomagnetically 分离的苏丹生命线行动在1DIV 表示未成熟标记.(A) 在相衬显微镜下显示双极性和三极形态。生命线表示 NG2 (B)、O4 (C) 和 O1 (D)。很少有星形胶质细胞存在于 1DIV (C, 红色), 鳞片酒吧 = 50 µm. 收集了3项独立实验的数据。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57543/57543fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了检验 immunomagnetically 分离细胞的抗原表型, 对细胞进行了 24 h (1DIV) 和 72 h (3DIV) 的免疫荧光染色。在 1DIV, 细胞似乎是双或 tripolar 在相对比显微镜下 (图 2A-B), 特征的早期增殖生命线行动 (OPCs 和或 preoligodendrocytes)。66.1, 8.4% 的这些细胞被 NG2-labeled (图 2B), 58% @ 9.4% 的细胞被 O4-labeled (图 2C)。O1 (GalC)-标记, 标记的更成熟的细胞在少突胶质谱系, 更不常见 (7.4%) 6.0% ,图 2D)。这个标记剖面表明, 大多数细胞在这一点上是预少突胶质与较小的百分比不成熟或成熟的寡核苷酸, 可能有一些少突胶质祖细胞 (OPCs)。星形胶质细胞, 如 GFAP 染色所证明的, 由 0.57 1.0% (图 2C) 组成。这些数据表明, 经改良的技术分离的少突胶质高度纯化, 多数表现模式为不成熟少突胶质的典型标记。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57543/57543fig3.jpg"> 图 3: 成熟的生命线标记在3DIV 的表达式.(A) 在相对比显微镜下的生命线显示更复杂的形态学。在3DIV 时, NG2 (B) 的表达式显著减少, 而 O4 (C) 和 O1 (D) 则增加。在 3DIV (C、红色)、缩放条 = 50 µm 中, 很少有星形胶质细胞可见. 数据是从3项独立实验中收集的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57543/57543fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在 3DIV, 少突胶质形态学比 1DIV(图 3A) 中的单元格显得更为复杂。此时, 与1DIV 相比, 早期少突胶质标记 NG2 的细胞阳性率要低得多. NG2-labeled 细胞由31.2 到 17.1% (与 66.1 @ 8.4% 在 1DIV ,t-测试 p < 0.0001 ,图 3B, 图 4A)。虽然在这里没有分析, 平均染色强度的细胞被认为是阳性的 NG2 是减少3DIV 相比, 1DIV。大多数单元格 (81.9% @ 4.884%,图 3C) 显示 O4-labeling, 它高于 1DIV (58% @ 9.4%, t-测试 p < 0.0001,图 4B)。此外, 几乎一半的细胞 (47.2% 16.4%,图 3D) 显示染色的 O1 (GalC), 一个标志更成熟的少突胶质血统, 表明细胞分化为一个更成熟的表型。与 1DIV (7.4% @ 6.0%、 t测试 p < 0.0001、图 4C) 相比, 3DIV O1+单元格的增加也很重要。此时, 星形胶质细胞的存在非常低 (0.8% 1.5%,图 3C), 与 1DIV (0.57% @ 1.0%, 不显著 (ns),图 4D) 相比, 它没有显著的差异。这些结果表明, 随着时间的推移, immunomagnetically 孤立少突胶质能够区分在体外到成熟的阶段三, 对其他细胞类型如星形细胞的污染极少。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57543/57543fig4.jpg"> 图 4: 在1DIV 和 3DIV (-d) 中对 OL 标记的表达式进行量化.在 1DIV, 66% 的细胞被 NG2+, 58% 的细胞被 O4+, 7% 的细胞被 O1+和0.5% 的细胞是 GFAP+。在 3DIV, 31% 的细胞被 NG2+, 81% 的细胞被 O4+, 47% 的细胞被 O1+和0.7% 的细胞是 GFAP+。这里提出的平均值, ** 表示 p < 0.0001, ns = 不显著。数据从3独立实验收集。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57543/57543fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes at JoVE.com

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

我们描述了主鼠标少突胶质的磁隔离, 它允许对体外区域性的细胞进行快速和特定的隔离。

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-specific-immunomagnetic-isolation-mouse-primary

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.5K 次观看.  State University of New York Downstate Medical Center. 我们描述了主鼠标少突胶质的磁隔离, 它允许对体外区域性的细胞进行快速和特定的隔离。

视频: Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

科研

神经科学

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

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

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

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. ...

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 of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

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 ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

共培养实验的奥里戈丁细胞和奥里戈丁酸基质的生成

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...