SS，联邦教育与研究部（BMBF 01 EO 0803），KB和欧盟委员会FP7格兰特PIRG08-GA-2010-276989，NEUREX，德国研究基金会资助SCHA 1442 /的Fazit基金会研究生奖学金的支持下3-1 CS有没有冲突的财务权益。

1。混合皮层细胞的分离和电镀混合皮层星形胶质细胞培养细胞分离，可以使用P1到P4鼠标幼崽。为了实现适当的星形胶质细胞的密度，它是必要的使用4鼠标小狗皮质每T75组织培养烧瓶中。因此，以下协议中的卷是计算一个单元使用4次鼠标幼崽的准备。 <ol><li>开始解剖过程之前，prewarm 30毫升的星形胶质细胞的培养基（DMEM，高葡萄糖+ 10％热灭活的牛胎儿血清+ 1％青霉素/链霉素;见表）至37℃。涂布一T75烧瓶中，用20毫升的聚-D-赖氨酸（PDL），在1小时，在细胞培养级的水的浓度为50微克/毫升，在37℃下在CO 2培养箱中。 </li><li>对于解剖过程中，准备所有必要的试剂和材料。你将需要:手术剪，抚平细小的镊子，平头镊子，纸巾，垃圾袋，70％乙醇D 2解剖菜（3.5厘米直径）冰填充用2 ml HBSS。 </li><li>轻轻握住，并用70％乙醇的小狗鼠标喷的头部和颈部。被牺牲的动物通过斩首使用的剪刀。 </li><li>执行正中切口，后到前，，沿头皮透露的头骨。 </li><li>切头盖骨仔细地从脖子到鼻子。两个额外的削减，以便进一步进入大脑进行:第一刀前部的嗅球，另一种劣质的小脑断开的头盖骨，颅底。 </li><li>颅翼片使用扁平喷嘴钳，轻轻翻转到侧和大脑被取出并置于第一解剖菜填充用HBSS。将菜上的冰和继续收集所有4个大脑。 </li><li>在立体显微镜下进行解剖过程的其余部分。首先，在嗅球和小脑使用精细除去解剖钳（ 图1B）。 </li><li>为了找回皮质，抓住后用细镊子的大脑，执行一个正中切口两半球，镊子插入第二组创建的树林和剥离的板状结构的皮质大脑。 </li><li>仔细解剖脑膜皮质半球拉用细镊子（ 图1D）。此步骤可避免污染最终的星形胶质细胞的培养由脑膜细胞和成纤维细胞。准备皮质半球转移到第二盘充满了HBSS，并返回到冰。所有4个皮质继续进行。 </li><li>最后，横切成小块，用锋利的刀片（约4至8倍）的皮质半球。 </li><li>在无菌条件下，转印的皮质片放入一个50毫升Falcon管中，并添加至总体积为22.5毫升的HBSS中。 </li><li>加入2.5毫升的2.5％的胰蛋白酶，混合孵育的组织在37℃下在水浴中30分钟。混合偶尔晃动每10分钟。 </li><li>离心5分钟，在300×g离心沉淀皮层组织片。 </li><li>仔细收集上清用倾析法。为了避免失去组织沉淀，您可以机械地保留使用吸管。解离成单细胞悬浮液，通过添加10毫升星形胶质细胞电镀介质和剧烈的移液使用10毫升塑料移液管，直到组织片解离成单细胞（20〜30次）的组织。调节音量使用星形胶质细胞电镀液20毫升。您可以证明的皮质组织的离解成单个细胞，用血细胞计数板计数。一个准备的4鼠标小狗的皮质产生10-15×10 6个分离的单个细胞。 </li><li>吸干PDL从T75培养瓶中，板的解离的单细胞悬浮液和孵育在37℃下在CO 2培养箱中。 </li></ol> 2。获取ENRiched星形胶质细胞文化<ol><li>改变介质的电镀混合皮层细胞和所有的3天之后的2天之后。 </li><li> 7〜8天，当星形胶质细胞的汇合和叠加的小胶质细胞上的星形胶质细胞层坐在暴露或已经脱离的星形胶质细胞层（ 图2）后，摇动的T75烧瓶中，在180rpm下30分钟以除去小胶质细胞上轨道摇床。弃上清含小神经胶质细胞或文化，如果你想和检查小胶质细胞，旋转和文化28,29板。 </li><li>添加20毫升新鲜星形胶质细胞的培养基中，并继续通过摇动烧瓶中，在240 rpm下6小时，以除去少突胶质细胞的前体细胞（OPC）。由于一些OPCs的不会完全从星形胶质细胞层分离，的继续用手剧烈振摇1分钟，为了防止OPC污染。弃上清或旋转板，如果你想培养OPCs的29。 </li><li>冲洗剩余的confluent的星形胶质细胞层两次，用PBS，吸出PBS，添加5毫升胰蛋白酶-EDTA和孵育在37℃下在CO 2培养箱中每5分钟检查支队的星形胶质细胞和执行支队的星形胶质细胞的击中烧瓶中对你的手的手掌（2-3倍）。 </li><li>后星形胶质细胞的分离培养瓶中，加入5毫升的星形胶质细胞的培养液中，自旋细胞在180×g离心5分钟，吸出上清液，加40毫升新鲜的星形胶质细胞的镀介质。一个T75细胞培养瓶中后的第一次细胞分裂产生约1×10 6个细胞。板细胞在CO 2培养箱中在37℃下在两个T75培养瓶中并孵育。更改的介质每2到3天。 </li><li> 12-14天，先拆后星形胶质细胞接种于合适的细胞浓度为24-48小时，然后再进行实验。一个T75细胞培养瓶中应该产生大约1.5-2×10 6个细胞后，第二次细胞分裂。 </li></ol>

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没有利益冲突的声明。

这里介绍的方法是基于从啮齿动物新生儿的大脑，于1980年27麦卡锡和德Vellis的原先所描述的星形胶质细胞培养制备。从出生P1到P4小鼠大脑皮质星形胶质细胞的分离和培养的方法，这里介绍的是速度快，产量纯星形胶质细胞是高度可重复性。这种技术可以很容易地转移到隔离来自其他物种的星形胶质细胞，如从大鼠或猪和由大脑的其他区域，如脊髓。而由麦卡锡和deVellis的方法，从新生儿脑星形胶质细胞祖细胞的分离产生高度增殖细胞，出生后的P1-P4鼠标幼崽，孤立的星形胶质细胞细胞的增殖和传播是有限的。分裂后星形胶质细胞在体外培养 7天（DIV），他们将增长至融合和成熟的。 在体内 ，最星形胶质细胞增殖，主要是完成P14 32。他再次，我们建议使用星形胶质细胞的实验，在每天21至28日的DIV（图2F），以确保孤立的星形胶质细胞的成熟型。由于星形胶质细胞培养增殖的内在约束不应该被劈裂的3倍以上。 在所描述的分离方法的关键步骤是皮质的消化和下面的研磨，消化的组织获得的单细胞悬浮液。因此，它是必要的，以优化研制后的大脑皮质组织为20-30倍，以得到单细胞悬浮液的胰蛋白酶的浓度和消化时间。为了减少变化的胰蛋白酶，应等分并应避免冻融循环。所描述的过程依赖于电镀混合皮层细胞PDL涂覆的培养瓶中，从而确保了对星形胶质细胞的结合和促进了合流的星形胶质细胞层电镀后几天。虽然PDL的涂层是没有必要的星形胶质细胞米aintenance之后所分离的小胶质细胞和少突胶质细胞，也可以进行一定的下游应用如免疫荧光染色。的第一小区分裂的良好的时序是非常重要的细胞的完整性和产量。如果星形胶质细胞的培养超越他们达到融合，你可能会失去大部分在第一个细胞分裂的细胞，由于没有足够的支队。这是不能克服脱离，越来越多的时间，因为大量的孵化时间与胰蛋白酶负面影响细胞的完整性。相反，几乎没有太皮质细胞悬浮液镀将导致汇合星形胶质细胞层的形成不充分。第一次细胞分裂的最佳时间是在7〜8天在电镀混合皮层细胞后，当星形胶质细胞的汇合和小胶质细胞坐在最上面的位置的星形胶质细胞层。 在皮质细胞培养，星形胶质细胞的异质性（&lt;强图3），因为它已被描述为星形胶质细胞在体内 33,34。但是，定义不同的星形胶质细胞形态和功能已经阻碍了数量有限的标记来识别和区分潜​​在的异质性星形胶质细胞亚型。成熟的纤维和星形胶质细胞反应性增生是一个很好的特点标记GFAP。但是，胶质纤维酸性蛋白（GFAP）几乎不表达成熟的原浆的星形胶质细胞，星形胶质细胞的标记物可用于所有限制其使用，它也表示由RG细胞在开发过程中，由B细胞在成人，作为一个阶段特异性的标记物，限制其使用。其他标记的星形胶质细胞，包括GLAST，ALDH1L1或BLBP，也由不成熟的星形胶质细胞中表达，因此，不完全成熟的星形胶质细胞的标记。最后，成熟的星形胶质细胞的标记物，如胶质纤维酸性蛋白，水通道蛋白-4，和S100B（ 图3）也越来越上调在出生后成熟。 在我们的手中，AST在4周龄的rocyte文化有成熟的星形胶质细胞在体内的特点。使用主星形胶质细胞培养，我们可以识别的分子机制如何出生的血液蛋白纤维蛋白原诱导星形胶质细胞活化17。我们的研究显示，纤维蛋白原是一种载体的休眠型TGF-β。导致活性型TGF-β的形成和激活TGF-β/Smad信号通路在星形胶质细胞17,26与纤维蛋白原的原代星形细胞治疗。这些结果已证实， 体内纤维蛋白原注射 。此外，星形胶质细胞控制其它类型的细胞所分泌的物质，它可以分析收获星形胶质细胞条件培养基，并应用此其他类型的细胞的条件培养基。我们以前使用的星形胶质细胞条件培养液中的纤维蛋白原治疗的星形胶质细胞条件培养液如何影响神经轴突生长的功能分析来分析。反应性星形胶质细胞表达和分泌蛋白质的的CSPG家庭，从而抑制神经轴突生长16。事实上，从处理过的纤维蛋白原的星形胶质细胞条件培养基显着下降突起长度和表示神经轴突生长17的细胞的百分比。 在本协议中所描述的星形胶质细胞的分离和培养的调查星形胶质细胞生物学提供了一个功能强大的工具，因为他们在不同的应用程序的可管理性，可大大体内完成调查。然而，应当牢记的已得到的星形胶质细胞在体外培养，并当它们反映许多星形胶质细胞的特性，它们也不同于在体内的星形胶质细胞。因此，其他的方法直接选择和隔离的星形胶质细胞通过immunopanning 35岁或抗体为基础的流式细胞术隔离36代表了新的途径，以进一步探讨星形胶质细胞的基本性质。

星形胶质细胞是中枢神经系统（CNS）中的一个非常丰富的细胞类型。在小鼠和大鼠皮质星形胶质细胞对神经元的比例是1:3，而每个神经元有1.4星形胶质细胞在人脑皮层1。在最近几年急剧增加星形胶质细胞功能的兴趣。一个关键功能的星形胶质细胞是神经元2,3结构和代谢支持的作用。新发现的星形胶质细胞的作用涵盖了广泛的功能。这些措施包括4-6在开发过程中，引导轴突和若干神经母细胞的迁移功能的突触传递，突触强度和信息处理的神经回路7-9，形成血脑屏障（BBB）的角色，在10和完整性11-13调节脑血管音14。的另一大特点是他们的星形胶质细胞对损伤的反应。在病理条件astrocyt的上课成为反应性和进一步上调表达的中间丝胶质纤维酸性蛋白（GFAP）和抑制细胞外基质（ECM）蛋白15,16。划定的健康组织的损伤部位形成胶质瘢痕，主要由星形胶质细胞分泌的细胞外基质蛋白，硫酸软骨素蛋白多糖（CSPG）家庭星形胶质细胞反应性增生，抑制轴突再生的主要因素，中枢神经系统损伤后15日至17日 。 星形胶质细胞在胚胎发育晚期和产后早期生命起源于放射状胶质细胞（RG）。规范已发生后，星形胶质细胞，星形胶质细胞的前体迁移到它们的最终位置，在那里他们开始终末分化过程中， 在体内 ，星形胶质细胞似乎是成熟的出生后三至四个星期，如所指示的典型形态18,19。 RG细胞亚群转化为脑室下区星形胶质细胞（B型细胞）。乙超视距，RG型和B细胞的功能在开发过程中的成人星形胶质细胞的神经干细胞（NS​​Cs）。喜欢的星形胶质细胞，RG和B型细胞也表达星形胶质细胞特异性的谷氨酸转运体（GLAST），脑脂质结合的蛋白（BLBP）的，和GFAP，表明这些标记可以不专门用于专门标记成人星形胶质细胞。相反成人的实质星形胶质细胞，没有分裂的健康的大脑，RG和B型细胞表现出干细胞的潜力，如自我更新的能力。星形胶质细胞失调有牵连的许多病症，包括老年痴呆症的20,21，亨廷顿氏病22，帕金森氏病23，Rett综合征24日和亚历山大的疾病25。此外，星形胶质细胞反应所有侮辱的中枢神经系统，导致星形胶质细胞活化和星形胶质细胞胶质瘢痕的形成16,26。星形胶质细胞胶质瘢痕形成以下大脑TRAUMA或脊髓损伤被认为是主要的屏障，防止神经细胞再生的15。 可靠的方法来隔离和保持纯化的细胞群的发展一直是我们了解神经系统的关键。创业的工作由麦卡锡和Vellis使调查人员，准备近纯培养的星形胶质细胞从新生大鼠组织27。关于使用这种方法，在这里稍加修改，以隔离小鼠脑皮层星形胶质细胞星形胶质细胞生物学已学到很多东西。补充体内研究中 ，星形胶质细胞以及空调介质使用所描述的体外培养，是宝贵的工具，以进一步深入了解星形胶质细胞的功能。

<table border="1"><tbody><tr><td> 工作解决方案的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td></td><td></td><td></td><td> 星形胶质细胞培养基 </td></tr><tr><td> DMEM，高糖</td><td> Life Technologies公司</td><td> 31966-021 </td><td></td></tr><tr><td> FBS，热灭活</td><td> Life Technologies公司</td><td> 10082-1​​47 </td><td>终浓度:10％ </td></tr><tr><td>青霉素 - 链霉素</td><td> Life Technologies公司</td><td> 15140-122 </td><td>最终浓度:1％ </td></tr><tr><td></td><td></td><td></td><td> 脑组织掏的解决方案estion </td></tr><tr><td> HBSS </td><td> Life Technologies公司</td><td> 14170-088 </td><td></td></tr><tr><td> 2.5％胰蛋白酶</td><td> Life Technologies公司</td><td> 15090-046 </td><td>终浓度为0.25％ </td></tr><tr><td></td><td></td><td></td><td> 其他 </td></tr><tr><td> 70％（体积/体积）乙醇</td><td>罗斯</td><td> 9065.2 </td><td></td></tr><tr><td>聚-D-赖氨酸</td><td>密理博</td><td> A-003-E </td><td> 50微克/毫升</td></tr><tr><td>水</td><td> PAA </td><td> S15-012 </td><td>细胞培养级</td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td>细胞培养级</td></tr><tr><td> 0.05％的尝试PSIN-EDTA </td><td> Life Technologies公司</td><td> 25300-062 </td><td></td></tr><tr><td> 0.45微米的无菌过滤器</td><td>赛多利斯</td><td> 16555 </td><td></td></tr><tr><td> 3.5厘米培养皿</td><td> BD猎鹰</td><td> 353001 </td><td></td></tr><tr><td> 15毫升的Falcon管</td><td> BD猎鹰</td><td> 352096 </td><td></td></tr><tr><td> 50毫升Falcon管</td><td> BD猎鹰</td><td> 352070 </td><td></td></tr><tr><td> 75平方厘米的组织培养瓶中</td><td> BD猎鹰</td><td> 353136 </td><td></td></tr><tr><td>钳，细</td><td>杜蒙</td><td> 2-1032，2-1033 </td><td> ＃3C＃5 </td></tr><tr><td>钳，平头</td><td> KLS马丁</td><td> 12-120-11 </td><td></td></tr><tr><td> 13厘米的手术剪刀</td><td>蛇牌</td><td> BC-140-R </td><td></td></tr><tr><td>体视显微镜</td><td>徕卡</td><td></td><td> MZ7.5 </td></tr><tr><td>体视显微镜+相机</td><td>徕卡</td><td></td><td> MZ16F; DFC320 </td></tr><tr><td>显微镜+相机</td><td>蔡司，佳能</td><td></td><td>的普里莫垂直的PowerShot A650 IS </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td>轨道振动筛</td><td> Thermo Scientific的</td><td> SHKE 4450-1CE </td><td> MAXQ 4450 </td></tr><tr><td>水浴</td><td> JULABO </td><td></td><td>小号W20 37°C; </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

星形胶质细胞是哺乳动物大脑中的细胞类型丰富，但仍有许多工作有待了解它们的分子和功能特性。星形胶质细胞在体外培养系统可以用来详细研究这些神经胶质细胞的生物学功能。此视频协议显示了如何获得纯鼠标幼崽的混合皮层细胞的分离和培养的星形胶质细胞。该方法是基于上没有存活的神经元，星形胶质细胞，少突胶质细胞，小胶质细胞，三个主要的神经胶质细胞种群的中枢神经系统的，在培养和分离。在第一天的文化代表图像显示的混合细胞群的存在，并表示时间点，当星形胶质细胞的汇合和小胶质细胞和少突胶质细胞应分开。此外，我们表现出纯度和完善培养的星形胶质细胞，用免疫细胞化学染色星形胶质细胞形态和新近被描述的星形胶质细胞的标记。这种文化系统可以很容易地获得纯星形胶质细胞和星形胶质细胞条件培养液对星形胶质细胞生物学研究的各个方面。

当完整的小鼠脑组织（ 图1A），小脑和嗅球必须要除去（ 图1B）的隔离。小心地取出（ 图1E）的小鼠脑干个别皮层（ 图1D）（图1C）和脑膜皮层剥离。脑膜的是明显的由脑膜动脉系统的和不完整的去除污染由脑膜细胞和成纤维细胞，星形胶质细胞的最终文化结果。 电镀混合皮层细胞悬浮液后，某些星形胶质细胞已经附加到培养烧瓶中，在24小时内（ 图2A）。然而，细胞培养物上清液，将包含细胞碎片和死亡的神经元的第3〜5天（ 图2A-C），作为培养基中有利于神经胶质细胞的存活和生长的。一旦星形胶质细胞的汇合和小胶质细胞坐在的astrocyt暴露E层，通常在7天隔离后的混合皮层细胞，星形胶质细胞被分离的小胶质细胞和原花色素振荡（ 图2D）。 2天之后的第一次分裂细胞星形胶质细胞形态。在这一点上的星形胶质细胞的密度是低的胶质细胞和星形胶质细胞是浩繁（ 图2E，黑色箭头标记一个单元格）。在这一点上的预期收益率较低，约4-6×10 5个细胞T75细胞培养瓶中。然而，细胞增殖和成熟的文化在这个时间点。的星形胶质细胞通常是用于实验的第21天到第28（ 图2F），以确保一个成熟的孤立的星形胶质细胞的表型。在这一点上的预期的收率是约1.5-2×10 6个细胞每T75组织培养烧瓶中。 星形胶质细胞培养纯度的特点，通过电子显微镜形态学研究，细胞标记物的表达和药物的反应27。了污染strocyte文化可以检查通过使用小胶质细胞标记（IBA-1）和原花色素（O4）。应检查所获得的星形胶质细胞培养的纯度和成熟的免疫细胞化学检测GFAP蛋白的抗体。据预计，大多数的细胞显示一个密集，丝状的信号（ 图3）。我们的隔离和皮层星形胶质细胞的培养方法，导致在星形胶质细胞的纯度大于98％，所揭示的对GFAP蛋白（ 图3）中使用的抗体。如果该过程成功，污染细胞是罕见的（&lt;2％），并含有小胶质细胞和原花色素，它可以通过使用IBA-1和O4分别染色。如果需要的话，进一步的分析，也可以使用其他的星形胶质细胞的标记物，如水通道蛋白4，BLBP，S100B和GLAST。此外，以GFAP，这些标记都表示，通过28天的星形胶质细胞培养。最近的基因表达纯化的星形胶质细胞群体的分析发现了新的潜在的ASTrocyte标记，如叶酸代谢酶ALDH1L1 30,31，这也是大多数分离的大鼠星形胶质细胞（ 图3）所表示。 <img alt="图1" src="/files/ftp_upload/50079/50079fig1.jpg" /> 图1。 A）全脑解剖产后（P3）鼠标皮质 。 B）搬迁后的嗅球及小脑脑。 C）的分离皮层剥离板状结构从大脑皮质。 D，D&#39;）皮层腹侧和背侧网站与脑膜（黑色箭头表示脑膜动脉）。 E）的Cortex没有脑膜。比例尺，1.5毫米。 <img alt="图2" src="/files/ftp_upload/50079/50079fig2.jpg" /> 图2。分离后的不同时间点孤立的混合皮层细胞和星形胶质细胞纯文化的形态概述。A）电镀混合皮层细胞后第1天。第一星形胶质细胞的附着到烧瓶底部（黑色箭头），死亡的神经元是在上清液中。 B）电镀混合皮层细胞后第3天。星形胶质细胞层正在形成（黑色箭头）。神经元是几乎不存在。 C）后5日内电镀混合皮层细胞。第一小胶质细胞和OPCs的顶部的星形胶质细胞层（黑色箭头）。 D）电镀混合皮层细胞后第7天。星形胶质细胞层完全融合的。 E）分裂后剧烈摇动，2天取出后小胶质细胞和原花色素，贴壁细胞，星形胶质细胞形态与低密度（箭头表示一个细胞）。 F）星形胶质细胞层示出高密度的第一分割后2个星期。比例尺，10微米。 <img alt="图3" src="/files/ftp_upload/50079/50079fig3.jpg" /> 图3。免疫的小鼠原代星形胶质细胞的尽头星形胶质细胞的主要文化的纯度。Tures的标记GFAP，GLAST，S100B，水通道蛋白4，ALDH1L1和BLBP（全部绿色）显示星形胶质细胞纯小学文化。细胞核染色，4&#39;，6&#39;-二脒基-2 - 苯基吲哚（DAPI）（蓝色）。比例尺:10微米。

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

星形胶质细胞被确认为是正常的大脑发育和功能，以及中枢神经系统的修复是必不可少的基本​​生物过程的多功能细胞参与。在这里，我们提出了一个快速的过程，以获得纯净的小鼠星形胶质细胞培养研究本类主要的中枢神经系统的细胞生物学。

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 ...

isolation-and-culture-of-mouse-cortical-astrocytes

Nanyang Technological University

Research

JoVE Journal

Neuroscience

91.1K 次观看.  University of Freiburg. 星形胶质细胞被确认为是正常的大脑发育和功能，以及中枢神经系统的修复是必不可少的基本​​生物过程的多功能细胞参与。在这里，我们提出了一个快速的过程，以获得纯净的小鼠星形胶质细胞培养研究本类主要的中枢神经系统的细胞生物学。

视频: Isolation and Culture of Mouse Cortical Astrocytes

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

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

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

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

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

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

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

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...