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统基于相机的显微镜技术。虽然显微镜已经在高通量筛选成功用于鉴定OPC分化诱导剂，数据采集是固有地慢，因为它需要数百图像​​捕获序列的编程，几个小时的数据采集，分析为th图像文件，以及用户定义的算法的ousands以区分正常化或分析18,19个别细胞体。此外，基于照相机的显微镜系统中，是不可行的从培养容器的整个表面获得的数据。相反，代表图像必须从单井中的不同区域，这可能导致位置偏差和假阳性命中被捕获。通过比较，该测定检测来自内的给定井的所有小区的MBP表达，和多块板可以同时进行扫描。虽然这种方法能够在96孔或384孔格式适于使用中，当正在测试更少治疗的24孔板可优选的。我们已观察到，在24孔板由于液体处理细胞损失被最小化，这是一个应设计高通量实验时，应考虑的并发症。 该测定的一个重要参数是细胞的密度differenti时通货膨胀是在日0开始培养太少细胞似乎更慢区分和死光，而高密度的文化表现自然分化。这两种情况下可能会降低该测定的动态范围和可靠性。为了实现细胞的健康和细胞密度之间的最佳平衡，我们的文化OPCs的3天不新鲜PDGF-AA的媒体交换或补充。我们推测，由于PDGF-AA的在体外 （第0天），为蜂窝分解代谢的结果，通过3天的介质下降的浓度，细胞的增殖速率将减慢，使得扩散通过PDGF-的第一天降低AA撤出。这种技术最好当细胞电镀过程中均匀分布的。吹打细胞沿着井的侧面，用图8个运动应促进均匀分布将它们混合。移液入井的中心趋向于产生围绕的边缘细胞簇以及少呃细胞附着到中心。这种现象可能是由于由液体吸移力新鲜干燥的PLL层的破坏。在周边细胞堆积可能会减少，因为密度依赖分化检测灵敏度。要注意，在肌动蛋白/ Olig2的信号的强度显著差异药物处理和控制，PDGF戒断文化之间发生是很重要的。显著减少肌动蛋白/ Olig2的信号可能预示药物毒性，而显著增加肌动蛋白/ Olig2的信号可能表明药物诱导的增殖。我们通常从促分化药物处理的细胞相比，PDGF撤出控制观察略高肌动蛋白/ Olig2的信号。我们认为这差异的PDGF撤离集团在4天的分化过程率较高的自发凋亡。在评估oligodendrogenesis，MBP正常化肌动蛋白/ Olig2的应该纠正为细胞数量的差异。为了评估TOXI城市和利用该测定增殖，抗MBP抗体可以针对适当的标记的初级抗体进行交换。 当实验需要的定量和定性评估，以限制固定大鼠少突胶质细胞培养物来的曝光是重要的Triton用4％PFA固定细胞，当X-100。其他免疫细胞化学协议少突胶质细胞培养物，在两者的抗体孵育步骤通常包括的Triton X-100，以提高抗体的表位可访问性，可根据实验要求成功地使用。另一方面，虽然MBP是胞内蛋白质有可能排除的Triton X-100完全如果替代固定方法是采用能充分透化细胞。使用此处所描述的条件下，我们观察到的Triton X-100将影响少突胶质细胞的观察形态学以及MBP染色的定位当以高浓度使用。我们看不出抗体染色一个显著下跌时从抗体孵育步骤省略的Triton X-100。作为抗体孵育期间在图6中所示 ，包括0.4％的Triton X-100中的透和阻断步骤，和0.1％的Triton X-100，减少了在不连续的MBP染色髓鞘过程和结果的检测。因此，对于定量和定性评估它可能是足够的此处所述使用的Triton X-100的浓度较低。 而从大鼠使用A2B5选定的OPCs可能是有利的，我们观察到用A2B5选定的OPCs从C57BL / 6小鼠中的双红外线荧光扫描（DIFS）测定可比较的结果，尽管每小鼠细胞产率通常多较低（数据未示出）。了OPC纯化其它协议也可以是与该测定兼容。例如，少突胶质细胞标记O4可以用作抗原以纯化INT使用磁珠20 ermediate阶段少突胶质细胞前体。作为替代基于磁性的方法，OPCs能由高速轨道振荡和差的粘合方法21包含星形胶质细胞和小胶质细胞9天旧混合胶质细胞培养物中富集。此外，免疫淘洗其中纳入如果文化纯度优于高细胞产率22可用于正面和负面的小区选择过程。除了研究如何治疗剂可直接提升在纯培养的OPCs的分化，有时需要考虑通过其他细胞类型在共培养系统的药物的间接影响。在MS，自身反应性T效应细胞的浸润进入CNS被认为在疾病进展23的作用。效应T细胞可以分泌可能抑制OPCs的分化在脱髓鞘部位促炎因子。因此，感兴趣的识别尊者peutics可保护的OPCs和阻止加剧损伤这些促炎因子。在DIFS分析可能是理想的T细胞生长在悬挂和可以洗涤从少突胶质细胞培养MBP量化之前离开造型这项活动。对于涉及贴壁细胞如神经元，星形胶质细胞，小胶质细胞和共培养研究中，Olig2的正常化也可以采用以收集从具体的少突胶质细胞的数据。因此，DIFS测定的灵活性可以便于各种实验设计的。 总之，这个协议提供了快速和可靠的方法来量化A2B5阳性大鼠的OPCs 体外的oligodendrogenesis。这种隔离方法一直致力于提供可行的OPCs是响应于既定分化线索的产量高。可在多井格式的船只采用多种实验范式，它扩展了该系统MA的效用进行敏感性分析NY不同的应用。此外，DIFS测定可适应于高通量药物筛选，或可被用来验证低通量测定，例如定量PCR和Western印迹的结果。重要的是，该系统可以提供用于脱髓鞘疾病如多发性硬化的识别OPC靶向治疗的一种简单而有效的手段。

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 border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:691px;">Dissection Materials</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PBS without Ca2+ and Mg2+</td>
			<td>Mediatech</td>
			<td>21-040-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Polystyrene Petri Dishes</td>
			<td>Fisherbrand</td>
			<td>0857512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Dissection Microscope</td>
			<td>Motic</td>
			<td>SM2-168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Dissociation Materials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>Dissociation Tube Rotator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neural Tissue Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td>Enzymatic Dissociation Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#956;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">A2B5 Column Purification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-A2B5 Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-864</td>
			<td>Bead Bound A2B5 Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Magnetic Selection Columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Corning</td>
			<td>46-034-Cl</td>
			<td>2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>0.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDGF-AA</td>
			<td>PeproTech Inc</td>
			<td>100-13A</td>
			<td>20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Hybri-Max 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodothyronine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td>45 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clemastine fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>SML0445-100MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quetiapine hemifumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>Q3638-10MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td>60 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1524</td>
			<td>10 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrecine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>16 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>40 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0135</td>
			<td>50 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">d-Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apo-Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4161</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trace Elements B</td>
			<td>Corning</td>
			<td>25-022-Cl</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Black Visiplate with lid</td>
			<td>Perkin Elmer</td>
			<td>1450-605</td>
			<td>Tissue culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Immunocytochemistry and Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>100-13A</td>
			<td>4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>78787</td>
			<td>0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Jackson Immuno Research</td>
			<td>005-000-121</td>
			<td>5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-MBP</td>
			<td>Biolegend</td>
			<td>SMI-99P</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Actin</td>
			<td>Santa Cruz Biotecnology</td>
			<td>SC-7210</td>
			<td>1:200 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Olig2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse 680RD</td>
			<td>Licor</td>
			<td>926-68070</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit 800CW</td>
			<td>Licor</td>
			<td>926-32211</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-mouse</td>
			<td>Life Technologies</td>
			<td>A11032</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold antifade with DAPI</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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）中删除。隔离工艺有效地消除了脑膜和成纤维细胞，但是当脑膜和血管被去除（图1E）酶消化得到改善。最后，为1毫米×1毫米的组织块被用于随后的组织离解的步骤制备（图URE 1F）。 良好的OPC隔离应导致最后的旋转步骤（图2A）以后一个紧凑细胞沉淀。一旦镀显微镜（图2B）下观看时，这些培养物会相对自由细胞碎片。次优隔离可能导致大量蓬松粒料由于大量的细胞碎片的存在。这些碎片会强烈粘附于PLL的涂覆容器中，并可以与培 ​​养物（ 图2C）干扰。如果一个大的和柔软的沉淀物化，它可能有助于悬浮细胞在10ml的PBS中，并在&lt;300×g的重复最后的旋转10分钟。一旦隔离完成后，培养物的纯度可以通过流式细胞术进行评估，以确定A2B5阳性细胞的百分比。一个成功的隔离应该导致那些&gt; 95％为阳性细胞A2B5一池（图2D - F）。相比用荧光染料缀合的抗A2B5抗体染色的细胞，未染色细胞应显示为人口负，并且可以用作门控的基线参考。此外，我们以前曾表明使用A2B5抗原产量的文化，通过免疫细胞化学染色11阳性PDGFRα的OPCs的选择。 这表现在免疫细胞化学，在治疗开始时（0天）的OPCs是Olig2的+ / MBP -而细胞是Olig2的+ / MBP由第4天+（图3）。在第0天及4天的OPCs和少突胶质细胞的最优汇合示于代表亮场图像（图4A）。在第0天的情况下的MBP表达充当定量oligodendrogenesis的基线。自发分化为PDGF-AA戒断的结果作为阴性对照，而45纳米甲状腺激素（triiodothyronine; T3）可以用来作为阳性对照11,15。喹硫平，也称为思瑞康，和氯马斯汀，也被称为Tavist，被FDA批准，已被证明对加速体外 oligodendrogenesis和可被用作附加阳性对照16,17药物。 图4B示出有代表性的双通道红外线荧光扫描表明为每个一式三份这些条件下，预期的结果。这样高度差异化的文化出现在合并后的图像黄肌动蛋白和MBP信号进行伪彩色绿色和红色分别。结果表明，在所述PDGF撤出条件的MBP表达在第4天为4.5±1.4倍高，相对于0天，而在MBP的倍数变化由于与甲状腺激素，喹硫平，和氯马斯汀治疗为33.9±4.1，33.4±1.0和32.8±0.5分别（图4C）。测定的鲁棒性和准确性由SM证明该三份样品，这是约20个标准差PDGF撤出控制上面的大治疗激活窗口中的所有标准偏差。 而肌动蛋白用作在少突胶质细胞培养物归一可靠的参考基因，也可使用替代的参考基因。 OLIG2是在少突胶质细胞谱系的细胞组成型表达一个核转录因子。其具体和组成型表达模式因此可以提供在异质文化的情况更优选的正常化的策略。 图5示出了在用10nM甲状腺激素分化的OPC培养，即使用时在表达相对MBP到PDGF撤出计算倍数变化是相同的OLIG2或肌动蛋白的正常化。据推测，由于在该实验中的OPC培养物非常纯净，这两种蛋白可以可靠地用于标准化使用。但是，我们ofte由4 N天观察Olig2的阴性细胞的一个小口，但是似乎该人口向Olig2的阳性人群的比不所述PDGF撤出和T3处理组之间显著不同。因此，倍数变化不受影响。给定一个场景，一个处理可能导致Olig2的阴性细胞类型的增殖，规范化抗体的选择应慎重考虑。 <img alt="图1" src="/files/ftp_upload/53764/53764fig1.jpg" /> 图1:。鼠脑解剖过程中的解剖过程的逐步描述<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53764/53764fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53764/53764fig2.jpg" /> 图2:流式细胞仪孤立的分析OPCs的（A）磁性柱纯化后，将结合的细胞群体被塞入出列并离心，以形成一个紧凑的颗粒。碎片的量由粒料的大小来表示。 （B）若粒料紧凑，则碎屑将在培养最小。 （三）大型，蓬松颗粒通常会导致与大量碎屑的文化。培养物的纯度通过流式细胞术使用缀合APC的大鼠抗小鼠A2B5抗体来确定。 （D）的死细胞和碎片被从分析中排除所示前向散射和侧向散射图。 （E）的A2B5阳性群体可以通过使用未染色取样作为用于浇注的基线参考来确定。 （F）中成功分离的OPC应&gt; 95％阳性，A2B5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53764/53764fig2large.jpg" target="_blank">请点击此处争夺</a>WA更大的版本这个数字。 <img alt="图3" src="/files/ftp_upload/53764/53764fig3.jpg" /> 图3:OPC培养和分化过程中的双红外线荧光扫描（DIFS）试验用新鲜isolatedA2B5阳性大鼠OPCs的开始铺板于PLL的涂布培养容器中（第3天）。这些培养物增殖3天在20毫微克/毫升PDGF-AA的存在下，然后与第0天治疗媒体在新鲜的PDGF - 自由媒体实验因素处理在第2天完全补充，并且将细胞固定用DAPI（蓝色），为核反染色，在0天和第4天的代表性的培养物中进行的第4天免疫细胞化学对Olig2的（绿色）和MBP（红色）4％多聚甲醛。这些文化表现出油锅少突胶质细胞谱系细胞标记物Olig2的组成染色在天0和4，而染色的成熟少突胶质细胞标记物MBP只有EV通过的ident第4天<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53764/53764fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53764/53764fig4.jpg" /> 图4:使用双红外荧光扫描（DIFS）测定MBP定量 （A）在第0天的OPC培养物的密度和4天以获得最佳检测性能要求。 （B）的代表性的双红外线荧光扫描。 OPCs的是在24孔板中45纳米T3中，1μM喹硫平，1μM的氯马斯汀，或以一式三份的0.1％DMSO存在分化4天。含有的OPC一个单独的板块是固定0（+ PDGF）当天在并行进行了处理。肌动蛋白（伪彩色绿色）和MBP（伪彩色红色），使用LICOR奥德赛扫描器设置成检测700纳米和800纳米的排放量同时定量。 （ <stroNG&gt; C）MBP信号正常化肌动蛋白信号，结果被缩放到日0 + PDGF控制。使用GraphPad Prism 6.平均相对荧光单位±SD绘制<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53764/53764fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53764/53764fig5.jpg" /> 图5:Olig2的是用于在少突胶质细胞正常化的MBP表达可靠的参考蛋白质的OPCs一式三份分化在10nM的T3或0.1％DMSO溶媒对照的存在下，在24孔板4天。使用抗Olig2的或抗肌动蛋白的初级抗体对正常化进行双红外荧光扫描测定。 OLIG2 /肌动蛋白和MBP使用LICOR奥德赛扫描仪设置为检测700 nm和800 nm的排放量同时量化。 MBP表达无rmalized要么Olig2的肌动蛋白或表达，结果被缩放到0.1％DMSO控制。的平均相对荧光单位±SD均使用GraphPad Prism所绘6.结果表明归到两个参考蛋白的当计算出的倍数变化是相同的。 <img alt="图6" src="/files/ftp_upload/53764/53764fig6.jpg" /> 图6:的Triton X-100上的MBP染色效果的OPCs在24孔板中分化为在45纳米T3或0.1％DMSO存在4天。然后将培养物用4％PFA和染色Olig2的和MBP使用两种不同的免疫细胞化学的协议。对于高的Triton X-100的样品，将细胞透化1小时用0.4％的Triton X -100和0.1％的Triton X-100的存在下进行所有抗体孵育步骤。对于低的Triton X-100的样品，将细胞透化1小时用0.1％的Triton X-100，而antiboDY孵化不含Triton X-100进行。 OLIG2和MBP进行可视化用荧光染料标记二级和图像用一个基于摄像头的荧光显微镜获得的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53764/53764fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

交付<em&gt;在体内</em&gt;急性间歇性缺氧新生儿鼠害总理下区源性神经祖细胞培养

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...