授予赞助商:美国国立卫生研究院;授权号:R37 NS041435。

涉及受试动物的程序已通过机构动物护理和使用委员会（IACUC）和医学的约翰霍普金斯学院。 1.检测板，股票解决方案，和OPC基本媒体的制备注:此处描述的大鼠OPC基地（佐藤）媒体已经从先前公布的研究得出12,13。替代媒体制剂也可以是与此过程相兼容。 <ol><li>悬浮聚-L-赖氨酸（PLL），以在无菌去离子水中10微克/ ml的浓度。吸移管400微升稀释的PLL成黑色壁，透明底的24孔组织培养级培养皿的每个孔中。 </li><li>在37℃组织培养箱中或在4℃冰箱中过夜2小时外套组织培养皿。 </li><li>吸PLL从涂层菜至少20分钟前电镀。允许通过将24孔板与盖部分微开在组织培养的其余PLL来从井干引擎盖。验证井电镀孤立的OPC之前完全干燥。细胞不会粘附到具有液体PLL目前塑料。 </li><li> 制备 N-乙酰基-L-半胱氨酸（NAC）原液（1000倍）:在20ml Dulbecco改良的Eagle培养基（DMEM）的溶解100 毫克 N-乙酰基-L-半胱氨酸（NAC）。分装和储存在-20°C。 </li><li>制备氢化可的松原液（1000倍）:1毫升乙醇添加到1毫克氢化可的松和漩涡溶解。添加19毫升DMEM中，在-20℃搅拌该溶液中，等分试样并储存。加入氢化可的松到基础介质的已被显示增强神经胶质细胞14的存活。 </li><li>制备D-生物素原液（5000倍）:将2.5毫克d-生物素的50ml PBS中。加入0.1N NaOH中的1-2×5微升滴在溶解的作用。分装和储存在-20°C。 </li><li>制备胰岛素原液（100×）:在50毫升的组织培养级水中溶解25mg的胰岛素。加入250微升Ø˚F的1N HCl并混合直到溶液澄清。用0.22微米的过滤器，并储存于4℃下长达6周消毒。 </li><li>制备佐藤原液（100×）: <ol><li>制备孕酮原液（25微克/微升）:在100μl的乙醇溶解2.5毫克孕酮。 </li><li>准备亚硒酸钠储备液（400纳克/微升）:在100微升的0.1N氢氧化钠溶解4毫克亚硒酸钠。加入10 mL的DMEM混匀。 </li><li>向100ml的DMEM，补充1克人脱辅基转铁蛋白，1克牛血清清蛋白，和160毫克的腐胺和混合到完全溶解。加入25微升黄体酮原液，1000微升亚硒酸钠原液，混匀。分装和储存在-20°C。 </li></ol></li><li>制备的OPC基媒体:每100毫升的DMEM（4.5 g / L的D-葡萄糖，L-谷氨酰胺，和110毫克/升丙酮酸钠），加入100微升的NAC，加入100μl氢化可的松，20微升D-生物素，将1ml胰岛素1毫升佐藤股票，100微升微量元素B，2毫升的B-27补充物（50×），和1ml青霉素/链霉素（100倍）的。用0.22微米的过滤器，并储存在4℃下灭菌。 </li><li>制备PDGF-AA储液（1000倍）:将250微克12.5毫升的PBS与0.1％牛血清白蛋白（BSA），以使20微克/毫升的溶液。分装和储存在-80℃。 </li><li>准备OPC增殖媒体:OPC基地媒体辅以20毫微克/毫升PDGF-AA。 </li><li>准备OPC分化培养基:辅以45纳米碘甲状腺原氨酸OPC基地的媒体。 </li><li>制备柱缓冲液:向500毫升的PBS中，添加2.5克的BSA和2的0.5M乙二胺四乙酸中的溶液（EDTA）和调节pH值至7.2。用0.22微米的过滤器，并储存在4℃下灭菌。 </li></ol> 2.大鼠脑解剖注:P5-P7幼鼠在本协议所使用。各大鼠小狗皮质产生约1.5-2.0×10 6 A2B5阳性细胞。 <ol><li>消毒使用250的所有清扫设备6;℃加热珠灭菌。 </li><li>加15-20毫升的PBS（不含Ca 2+和Mg 2+）到50毫升锥形管中。一个50ml锥形管中是足够3-4大鼠小狗皮层。将50ml锥形管冰。使用50ml锥形管清扫期间举行切块皮层组织。 </li><li>加入冷PBS到10cm培养皿。这道菜将在光学显微镜下可用于解剖。 </li><li>通过与大型手术剪刀斩首牺牲幼鼠。喷雾头，用70％的乙醇。 </li><li>提取全脑<ol><li>用小剪刀切开皮肤下的中线和耳朵和剥离皮瓣背后。切开头盖骨下来中线从脑干开始到结束的眼睛。小心不要太过严厉，以维护底层皮层的结构。 </li><li>使两侧切口插入小型手术剪入枕骨大孔逊色于小脑。使眼睛之间的附加切口，蚂蚁erior至嗅球。 </li><li>小心剥离颅骨各占一半回来。在10cm培养皿填充有冷PBS取出全脑和地点。 </li></ol></li><li>根据解剖光镜下取出嗅球和小脑用细镊子手术。 </li><li>翻转大脑以便腹面是在光学显微镜下可见的。 </li><li>用细镊子直通过将皮质和下丘脑之间封闭的镊子技巧的深度关于大脑的2/3进行钝性分离。一旦镊子到位允许的端部打开。重复其他半球。 </li><li>挑逗下丘脑和中脑区皮层。 </li><li>通过保持脑在其后表面和剥离它朝向脑的前端去掉下丘脑，丘脑，中脑。切断前的连接。 </li><li>向外洪亮地传颂着，然后切断其连接删除海马皮质用细弯夹层镊子。 </li><li>用细弯夹层镊子向外斜向运动从底层皮质再杀它删除剩余纹状体。 </li><li>清除任何血管和脑膜从皮质腹面。 </li><li>翻转大脑使得背表面是可见的。脑膜和血管应当是显而易见的。用细镊子剥离从底层皮层剥离脑膜。开始从嗅球连接位置剥离。 </li><li>总共3-4幼鼠完整步骤2.4-2.14。 </li><li>放置3-4解剖鼠小狗皮质成干培养皿用无菌刀片切碎直至1毫米×1mm的块被实现。通过从一个50ml锥形管（第2步），然后放回到冰上用PBS漂洗菜收集组织。 </li></ol> 3.酶和机械组织解离注意:对于这个步骤，建议使用基于木瓜蛋白酶解离的神经套件。使用氖乌拉尔解离试剂盒（P）时，这是最适合的OPC隔离特殊步骤进行说明。 <ol><li>通过稀释酶P与适当的缓冲液制备第一离解酶。每50ml锥形（1样品）中的内容将与总的1,950微升酶混合物的再悬浮。地点在一个37℃浴中的酶混合物10分钟。 1样品:1900微升缓冲+ 50微升木瓜酶</li><li>自旋所有50ml含3-4切丁大鼠小狗皮质锥形管在300×g离心3分钟。 </li><li>吸出上清液，并添加1,950微升酶溶液，以每个样品管中，颠倒试管并轻轻晃动掰开沉淀。 </li><li>孵育在37℃组织培养孵化用于与连续的管旋转15分钟。 </li><li>在过去的5分钟的孵化，准备第二酶混合物A.稀释在适当的缓冲酶。共30微升将被添加以每次50ml锥形样品管中。 1样品:20微升缓冲液+10μl的酶的</li><li>一旦温育完成添加30微升第二酶组合，以每管并颠倒混合。 </li><li>使用连接到吸移管控制器的玻璃巴斯德移液管，机械吹打10-15倍或直到组织片尺寸减小，并且溶液变得混浊离解的组织。返回样品到37℃组织培养孵化与连续旋转15分钟。 </li><li>使用1毫升吸管吸取各组织匀浆样品10-15次。 </li><li>用200微升吸管吸取各组织匀浆样品15-20倍</li><li>返回所有样品至37℃组织培养孵化10分钟以连续旋转。 </li><li>加入10毫升的PBS（以钙离子和镁离子）向每个样品和过滤匀浆通过放置在一个50微米的40微米的微孔过滤升管。用另外1-2毫升的PBS冲洗过滤器。 </li><li>池中的所有样品匀浆成1个50毫升的锥形管，并获得总细胞数。 </li><li>在进行细胞计数后，平分匀浆成的15毫升锥形管中（1管每个样品）。离心机在300×g的10-12分钟。 </li></ol> 4.抗A2B5珠应用，柱纯化，以及电镀<ol><li>执行列缓冲器和抗A2B5微珠计算。加7微升柱缓冲液为每1×10 6个细胞。添加2微升反A2B5微珠为每1×10 6个细胞。 </li><li>在300 xg离心重悬细胞以10ml柱缓冲液和离心机的总体积为10〜12分钟，结合所有样品。 </li><li>重悬在柱缓冲液适量细胞沉淀如在步骤4.1计算。如果颗粒是大的和松散，只添加柱缓冲液的体积的约一半，以保持反体在细胞再悬浮在适当的浓度。 </li><li>孵育细胞悬浮液在4℃10分钟。 </li><li>添加抗A2B5微珠（在步骤4.1计算的），颠倒几次，并在4℃下孵育30-60分钟。轻轻颠倒管每10分钟几次。较长的孵育时间可能会增加细胞产量，但会增加非特异性结合。 </li><li>加入5倍体积的柱缓冲。如果将细胞再悬浮于1毫升的体积，加入5 ml柱缓冲液中，而如果再悬浮于2ml的细胞，加入10 ml柱缓冲液中。 </li><li>离心机在300×g下10分钟。 </li><li>确定多少LS柱将需要的纯化。其中LS柱足以15-20×10 6个细胞。 </li><li>重悬在1000微升每LS柱的柱缓冲液的细胞沉淀被使用。 </li><li>素通过加入5ml柱缓冲液的各LS柱。收集通过在50ml锥形管中的流动。一旦该列卜FFER已完全通过上部腔室通过，应用1000微升的细胞悬液到每一列和允许细胞通过重力运行。 注意:如果细胞的密度太高，则该列可能运行缓慢。 </li><li>细胞悬浮液已通过该柱后，立即缓慢3毫升柱缓冲液添加到每个列以洗涤未结合的细胞。如果洗涤容易通过塔（1滴为每30-45秒）运行时，用3ml柱缓冲液洗两次。 </li><li>删除每一列，并将其放置在一个15毫升的锥形管。加入5 ml柱缓冲液中，并在使用该包装与该列的塑料柱塞以很快的速度扎通过该柱的缓冲液中。插铣会打跑结合的细胞从柱释放他们。 </li><li>通过第二轮LS列运行示例增加纯度。使用第一遍期间使用列数的三分之一。素用5ml柱缓冲液中的列。允许柱缓冲液穿过上部腔室。 </li><li>自细胞悬浮液的体积会大得多，均匀涂抹1000微升细胞悬液，以每列，并且允许该悬浮液用额外的1000微升补充之前通过该柱的上部腔室通过。 </li><li>一旦进入细胞悬浮液已完全施加到第二轮列，用3ml柱缓冲液洗2-3次。 </li><li>删除每一列，并将其放置在一个无菌的15毫升锥形管。加入5 ml柱缓冲液中，沉浸使用打包与列塑料柱塞缓冲区。插铣会打跑结合的细胞从柱释放他们。 </li><li>离心在300 xg离心所述细胞悬浮液为12-15分钟。小球应该出现紧凑。 </li><li>重悬细胞沉淀在5-10毫升预热的OPC扩散介质（辅以PDGF-AA 20毫微克/毫升的OPC基础媒体）的。 </li><li>算使用血球细胞。 </l我&gt; <li>板上的细胞。稀释细胞使用OPC扩散介质60,000个细胞/毫升。进入每个黑色，透明底24孔，吸管500微升沿井的侧细胞获得每孔30,000个细胞。通过水平摇动板采用八字形运动混合细胞。如果在48，96，或384孔板进行测定，添加15,000，7500或1500细胞每孔，分别。这些数字可以根据个人板的规格进行调整。 </li><li>准备一个单独的盘为0天，基线控制文化。这个控制板应用4％多聚甲醛如在第5.7节描述的，当分化在第0天开始。 </li><li>培养细胞中的OPC增殖培养基中于37℃下进行3天，没有媒体的变化。 </li></ol> 5.诱导分化OPC和固​​定用4％多聚甲醛注:当测试小分子对oligodendrogenesis的影响，我们经常dissolvÈ药物的二甲亚砜（DMSO）中，制备1000倍的股票，然后可以保存在-80℃，并直接稀释到佐藤媒体，以获得0.1％的DMSO的最终浓度。我们观察到在任一OPC增殖或分化用DMSO中该浓度没有改变（数据未显示）。 <ol><li>预暖的基础OPC媒体至37°C和解冻药物治疗股至室温。当在重复执行处理，在1.5ml管准备大约将1.25ml每个治疗组的介质。对于一式三份样品，使用2个毫升容量离心管占松弛。 </li><li>通过稀释处理股直接进入OPC基础媒体准备重复孔治疗预混液。简言之涡或轻轻拌匀每个mastermix，保证了治疗的均匀分布。 </li><li>制备45纳米甲状腺素（T 3）作为阳性对照和相应的车辆作为阴性对照。稀释10毫米T第3股1:1000于1ml的OPC基础介质中，然后在治疗主混合物进一步稀释如果需要降低的DMSO的最终浓度。 </li><li>吸从培养孔中一个治疗组的媒体在一个时间，以避免干燥的细胞。使用在玻璃巴斯德吸管的端部的200微升吸管尖以避免从井的侧面和向培养刮掉黑色材料。 </li><li>慢慢加入500μl治疗反应体系的沿井使用1毫升移液管的侧面。 </li><li>孵化培养所需的时间内，用新鲜的处理介质进行完整媒体交换每2-3天。后分化媒体4中的日子里我们经常观察到30-40％的MBP +文化。 </li><li>在所需的治疗期结束时，准备新鲜的4％多聚甲醛（PFA）或解冻冷冻的储存到室温。注意:PFA是剧毒的，必须在通风橱来制备。 </li><li>吸媒体，并慢慢加入4001，L的PFA到孔以固定细胞的侧面。将培养板在室温下20分钟。 </li><li>吸出PFA和慢慢加入500μl无菌的D-PBS（与钙离子和镁离子）的添加到井侧以洗涤细胞。重复洗涤共两次。不要吸出最后的清洗。 </li><li>包裹在封口膜和存储该板在4℃下用于以后的处理，或直接进入下一个步骤。板可以储存数周。 </li></ol> 6.免疫组化和定量髓鞘碱性蛋白注意:在24孔板进行液体处理程序时，推荐的工作速度快，以避免干燥的细胞。这里，建议使用多通道真空吸气器和多通道吸管的。 <ol><li>使板至室温，吸出孔中，并加入250微升的D-PBS（与钙离子和镁离子）的含有0.1％的Triton X-100和5％正常山羊血清（NGS）。在室温下孵育该板1小时，轻轻轨道振荡。 </li><li>通过稀释的小鼠单克隆抗-MBP抗体1制备主培养溶液:1000和兔多克隆抗肌动蛋白抗体1:含5％NGS 200中的D-PBS（与钙离子和镁离子）。可替代地，兔多克隆抗Olig2的以1:1000，可用于在混合胶质细胞培养少突胶质细胞特异性正常化。准备足够首要解决，以适应每孔250微升。 </li><li>在4℃孵育第一抗体过夜16-18小时轻轻轨道振荡。 </li><li>吸出主培养溶液中并在室温下洗涤细胞3×10分钟用500μlD-PBS（与钙离子和镁离子）的温和轨道振荡。 </li><li>而洗涤平板，通过稀释抗鼠680和抗兔800抗体1准备辅助孵化的解决方案:在D-PBS 500（有钙 </SUP&gt;和Mg 2+）含5％NGS。编制本解决方案时，最大限度地减少环境光曝光。 </li><li>吸出最后的清洗，并添加250微升二次孵化的解决方案。从光保护板并在室温下孵育其1小时轻轻轨道振荡。 </li><li>吸辅助孵化溶液并用500μlD-PBS（与钙离子和镁离子）的，在室温下，通过温和轨道振荡洗孔3×10分钟。 </li><li>扫描使用能够检测700纳米和800纳米的荧光发射的成像系统的板。作为一个起点，将焦点偏移3和敏感性1.5 MBP，5.0肌动蛋白和3.5 Olig2的。根据需要，以避免信号过度调整灵敏度值。 </li><li>量化oligodendrogenesis使用半自动化的基于软件的方法的程度。 <ol><li>使用该软件，设置板块分析模式，以"24井"，并调整围绕牛逼的圈子扫描的井，他外缘。将正常化通道"800"和出口总量和相对荧光强度为700纳米（MBP）和800纳米（Olig2的/肌动蛋白）通道。 </li><li>由强度在800nm​​处划分在700nm的强度，得到在相对荧光单位的归一化的MBP表达。计算重复测量的平均值，并根据需要进行了分析缩放表达到第0天+ PDGF控制。 </li></ol></li></ol>

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作者什么都没有透露。

这里介绍的协议描述了分离的大鼠的OPCs并响应实验因素体外 oligodendrogenesis定量了快速和可靠的方法。用积极的选择，可行的和纯粹的OPC的高收益直接用于实验目的。此方法简化了隔离，培养和分化的步骤到1周的时间帧，并固定细胞可以方便地储存在4℃下数周而不在测定灵敏度的任何损失。 与基于扫描器荧光测定法的一个主要优点是，数据采集和分析被大大加快与传?...

在哺乳动物中枢神经系统（CNS）高效神经传导需要通过少突胶质细胞轴突的髓鞘。在开发过程中，少突胶质细胞从被认为来自发展中国家前脑和神经管1的心室区迁移少突胶质细胞祖细胞（OPCs的）池出现。迁移后的OPCs分化为成熟的，少突胶质细胞髓鞘不仅方便高效的冲动传播，而且还提供与轴突营养支持2。成人CNS认为，分散在包括所有的细胞3约5-8％的灰质和白质的OPC充裕的人口。以下脱髓鞘损伤，OPCs的迁移到损伤部位，增殖和分化，以取代在暴露轴突丢失或损坏的髓鞘。然而，在一些疾病/损伤设置，这一过程被发现是低效率的，或者可以完全失败4。而慢性脱髓鞘被认为是增加性疾病，高效oligodendrogenesis和髓鞘再生的负担可能减轻症状5。因此它一直感兴趣的研究的OPCs 体外测定对oligodendrogenesis实验因素的影响。 洞察少突胶质细胞分化的不同阶段已经由阶段细胞特异性标记物的鉴定成为可能。自我更新的早期祖细胞可以通过（PDGFRα）血小板衍生的生长因子受体α的表达式定义，神经/神经胶质细胞抗原2（NG2）和A2B5 6 - 8。作为OPCs的启动其分化程序，并从细胞周期的撤回，它们下调这些标记物的表达，并开始表达指示premyelinating少突胶质细胞，包括环核苷酸3&#39;-磷酸二酯酶（CNPase）和O4的蛋白质。最后，它们分化成更成熟oligodendrocytes特征是髓鞘相关蛋白，包括髓鞘相关糖蛋白（MAG），蛋白脂质蛋白（PLP）和髓鞘碱性蛋白（MBP）的表达。 MBP是细胞内周膜蛋白和髓鞘的主要组成部分。缺乏MBP小鼠发展严重的表型在中枢神经系统髓鞘是显著下降导致震颤，抽搐和过早死亡9,10。在髓鞘形成的MBP这个重要作用，导致其作为在体外和体内 11少突胶质细胞分化的标记物使用。 可以使用多种不同的方法来实现的MBP的定量。 RT-PCR和Western blot分析允许在不同的治疗方案MBP水平的定量。免疫细胞化学是一种常见的定性方法，当使用基于相机的显微镜方法也可以是定量的。虽然这些系统是可靠的，根本少突胶质细胞分化的研究中，他们每个人都有自己的缺点限制了它在药物筛选使用。首先，所需的RT-PCR和Western印迹分析的主要的OPCs量减少，能同时被检查变量数。而进行免疫​​细胞化学电池的要求低得多，如果量化是目标相当长的时间，必须专门用于每次实验。众多的图像必须被捕获，然后量化，以方便实验分析。这些警告成为考虑需要高吞吐量评估研究的重要障碍。在这里，我们描述了利用来自免疫细胞化学和髓鞘定量Western印迹方法都基本方面的方法，而显著减少所需的细胞的数量和时间来完成定量分析。

<table><tbody><tr><td>&lt;strong&gt;Dissection Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech</td><td>21-040-CV</td><td>1x</td></tr><tr><td>10 cm Polystyrene Petri Dishes</td><td>Fisherbrand</td><td>0857512</td><td></td></tr><tr><td>Light Dissection Microscope</td><td>Motic</td><td>SM2-168</td><td></td></tr><tr><td>&lt;strong&gt;Dissociation Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>Dissociation Tube Rotator</td></tr><tr><td>Neural Tissue Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>130-092-628</td><td>Enzymatic Dissociation Kit</td></tr><tr><td>PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>20-030-CV</td><td>1x</td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>&lt;strong&gt;A2B5 Column Purification&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-A2B5 Microbeads</td><td>Miltenyi Biotec</td><td>130-097-864</td><td>Bead Bound A2B5 Antibody</td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Magnetic Selection Columns</td></tr><tr><td>EDTA</td><td>Corning</td><td>46-034-Cl</td><td>2 mM</td></tr><tr><td>PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>20-030-CV</td><td>1x</td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9647</td><td>0.50%</td></tr><tr><td>&lt;strong&gt;Culture Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PDGF-AA</td><td>PeproTech Inc</td><td>100-13A</td><td>20 ng/ml</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td>Hybri-Max 100 ml</td></tr><tr><td>Triiodothyronine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6397</td><td>45 nM</td></tr><tr><td>Clemastine fumarate salt</td><td>Sigma-Aldrich</td><td>SML0445-100MG</td><td>1 &amp;mu;M</td></tr><tr><td>Quetiapine hemifumarate salt</td><td>Sigma-Aldrich</td><td>Q3638-10MG</td><td>1 &amp;mu;M</td></tr><tr><td>N-acetyl cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>5 &amp;mu;g/ml</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>60 ng/ml</td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td>10 &amp;mu;g/ml</td></tr><tr><td>Putrecine</td><td>Sigma-Aldrich</td><td>P7505</td><td>16 &amp;mu;g/ml</td></tr><tr><td>Sodium Selenite</td><td>Sigma-Aldrich</td><td>S5261</td><td>40 ng/ml</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0135</td><td>50 ng/ml</td></tr><tr><td>d-Biotin</td><td>Sigma-Aldrich</td><td>B4639</td><td>10 ng/ml</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>5 &amp;mu;g/ml</td></tr><tr><td>Apo-Transferrin</td><td>Sigma-Aldrich</td><td>T1147</td><td>100 &amp;mu;g/ml</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4161</td><td>100 &amp;mu;g/ml</td></tr><tr><td>Trace Elements B</td><td>Corning</td><td>25-022-Cl</td><td>1x&amp;nbsp;</td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>17504044</td><td>1x&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>1x&amp;nbsp;</td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td>1x&amp;nbsp;</td></tr><tr><td>24-well Black Visiplate with lid</td><td>Perkin Elmer</td><td>1450-605</td><td>Tissue culture grade</td></tr><tr><td>&lt;strong&gt;Immunocytochemistry and Quantification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>100-13A</td><td>4%</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>78787</td><td>0.10%</td></tr><tr><td>Normal Goat Serum</td><td>Jackson Immuno Research</td><td>005-000-121</td><td>5%</td></tr><tr><td>mouse monoclonal anti-MBP</td><td>Biolegend</td><td>SMI-99P</td><td>1:1,000 Dilution</td></tr><tr><td>rabbit polyclonal anti-Actin</td><td>Santa Cruz Biotecnology</td><td>SC-7210</td><td>1:200 Dilution</td></tr><tr><td>rabbit polyclonal anti-Olig2</td><td>Millipore</td><td>AB9610</td><td>1:1,000 Dilution</td></tr><tr><td>goat anti-mouse 680RD</td><td>Licor</td><td>926-68070</td><td>1:500 Dilution</td></tr><tr><td>goat anti-rabbit 800CW</td><td>Licor</td><td>926-32211</td><td>1:500 Dilution</td></tr><tr><td>Alexa Fluor 594 goat anti-mouse</td><td>Life Technologies</td><td>A11032</td><td>1:1,000 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit</td><td>Life Technologies</td><td>A11008</td><td>1:1,000 dilution</td></tr><tr><td>Prolong Gold antifade with DAPI</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Licor Odyssey Clx Infared Imaging System</td><td>Licor</td><td></td><td></td></tr></tbody></table>

preparation of rat oligodendrocyte progenitor cultures and quantification of oligodendrogenesis using dual-infrared fluorescence scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

A2B5阳性的OPC使用磁性柱分离系统通过积极的小区选择隔离。在此之前的分离步骤，整个大脑从幼鼠是P5和P7之间除去。一旦整个大脑成功从头骨分离，嗅球和小脑使用精细外科镊子（图1A）中除去。为了分离大脑皮质组织下丘脑，丘脑和中脑被小心剥离（图1B）切除。接下来，海马和纹状体使用弯钳手术（ - D图 1C）<...

Watch this Scientific Journal Video about Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning at JoVE.com

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

大鼠少突胶质祖文化的制备和使用双红外荧光扫描Oligodendrogenesis的量化

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

preparation-rat-oligodendrocyte-progenitor-cultures-quantification

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.8K 次观看.  Johns Hopkins University, School of Medicine. Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

视频: Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

神经科学

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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 ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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 ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

用于神经干细胞和少突胶质细胞祖细胞分离的大鼠"脑挤奶"

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

作者聚焦:使用新型脑挤奶方案从活体动物中收集神经干细胞和祖细胞 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

大鼠少突胶质祖文化的制备和使用双红外荧光扫描Oligodendrogenesis的量化

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生产和使用慢病毒的有选择性地转导原少突胶质前体细胞

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

来自祖，人脑少突胶质细胞培养系统

小鼠初级少突胶质的快速特异磁分离

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

共培养实验的奥里戈丁细胞和奥里戈丁酸基质的生成

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法