我们要感谢艾莉森菲尔布鲁克，贝琳达Barbagallo和迈克尔·弗朗西斯对本文有见地的意见。研究报告本出版物是由R15区奖1R15NS070172-01A1授予​​M​​LL支持。

1，盖玻片准备:酸洗和烘烤<ol><li>至少提前2天清扫（步骤2），开始下面的步骤准备盖玻片。进行酸洗步骤以去除油脂，彻底清洁的盖玻片。 </li><li>负载盖玻片瓷支架的垂直位置盖玻片，防止它们相互接触。淹没支架和盖玻片放入盛有2M的盐酸（HCl）的玻璃容器中组织学。或者，如果瓷支架是无法使用，散布〜20盖玻片水平地覆盖100毫米的玻璃陪替氏培养皿的底部，并浸没在2 M盐酸盖玻片。 </li><li>置于玻璃容器（具有浸没在酸盖玻片）上摇杆表中通风柜并允许温和室温下摇动至少5小时。可替代地，酸洗涤盖玻片过夜在相同条件下。 </li><li>用蒸馏水H 2 O的（DH 2 O）通过转让持有的瓷器装洗盖玻片用盖玻片2 M盐酸以充满DH 2 O干净的玻璃容器中的组织学另外，倒出2 M盐酸的玻璃培养皿盖玻片，并加注DH 2 O。 </li><li>再次滗DH 2 O和加气站的玻璃容器用新鲜的DH 2 O洗重复共3快洗。 </li><li>通过返回的玻璃容器（含盖玻片和DH 2 O）摇臂上开始长的洗涤方案。允许温和搅拌，在室温下10-30分钟。然后，倒出DH 2 O和补充新鲜DH 2 O重复共计至少10次洗涤。另外，隔夜继续冲洗。 </li><li>放置洗涤盖玻片入炉预热到350℃。如果使用瓷支架，从DH移除保持器2 O-填充的玻璃容器，然后将用盖玻片夹持器直接入炉。如果使用的是玻璃培养皿，倒出DH 2 O和放置盖玻片用玻璃盘放入炉内ê。 </li><li>烘烤盖玻片至少五小时或过夜，在350℃。 </li><li>烘烤完成后，从细钳子瓷支架取出盖玻片，并放入无菌100毫米玻璃培养皿。离开盖玻片最初未放置在瓷座玻璃培养皿。 </li></ol> 2，鸡解剖和按病种付费的隔离<ol><li>删除肥沃，上演了从孵化器中（在37-38.5℃保持与温和的摇摆）白来航鸡的蛋。 注:以前的研究中使用7-10天胚胎的按病种付费2,8,11-13,16的隔离。 </li><li>发生在层流罩选择鸡蛋。进行在层流罩用的无菌溶液和仪器在无菌条件下在下面列出的所有步骤。在37℃水浴中预温的所有解决方案。 </li><li>裂纹蛋和地点的内容成塑料百毫米培养皿。使用标准的镊子胚胎移植到另外100毫米的培养皿。 </li><lI&gt;使用巴斯德吸管，加入温热PBS洗涤，培养皿，以保持组织湿润。按捏脖子用细镊子斩首胚胎。转印体到另一个干净的100毫米的培养皿。 </li><li>加入温暖F12HS10（火腿的F12，10毫米的HEPES，100单位/ ml青霉素，0.1毫克/毫升链霉素，10％胎牛血清）抛出与巴斯德吸管，以保持组织湿润。使用镊子在其背侧（ 图1A）定位胚胎。 </li><li>将含有胚胎组织上的层流罩双目立体视觉解剖显微镜百毫米塑料培养皿。用细镊子捏住发展到真皮层暴露心脏，肺等器官，使垂直正中切口，从尾巴到脖子。另外，进行一系列的小眼泪，露出内脏。 </li><li>轻轻地用2台无菌细镊子:1）掌握颈部和b）掌握主动脉或其他组织立即优于心脏和拉向尾部，取出内脏的动物。 </li><li>用巴斯德吸管来组织应用温水F12HS10。使用镊子，以去除任何剩余的心血管，呼吸或消化器官露出显影肋骨，脊柱和背根节（ 图1B-1D）。 </li><li>在解剖显微镜下，用细镊子沿腰脊柱，下到两侧肋骨，以进一步去除内脏和收集病种付费。通过从胚胎采摘完好病种付费病种付费领取。捏紧并切断了从到达对脊髓背根神经节和把握发展的神经到达从向周边反之亦然背根神经节的根部。 </li><li>将完好的DRG到35毫米的培养皿中充满了温暖的F12HS10。通过用细镊子夹持除去任​​何多余的组织，如背侧或腹侧根部可能已附着的DRG，从完整的DRG在35mm培养皿。 </li><li>为了获得病种付费优于腰部，取出胸宫颈癌放疗的腹侧半CAL脊柱。这是通过在显影脊柱的切口垂直于脊髓轴在上颈部区域和另一个切口在上部腰区进行。然后，使切口平行于沿右和脊柱的左右两侧的脊髓轴。从胚胎中，它暴露了暴露了脊髓（ 图1E，1F）提起脊柱的腹侧一半。 </li><li>为了能够轻松地访问病种付费，抓住脊髓用细镊子取出胸脊髓型颈椎病。使用镊子解除脊髓出了脊柱。作为近似，胸髓是在肋的水平和颈部区域优于肋。 </li><li>收集完整的胸椎和颈椎病种付费用细镊子，并将其放置在温暖的F12HS10与其他病种付费。除去可能附着病种付费多余的组织。 </li><li>冲洗了新的玻璃巴斯德吸管的内饰与F12HS10帮助防止病种付费粘吸管的墙壁。用移液管冲洗从小培养皿转移所有病种付费和F12HS10到无菌的15 mL锥形管，并立即进行第3步。 注:其他胚胎可以解剖，以增加病种付费的数量。的DRG应保持在F12HS10在37℃，直到准备步骤3。 </li></ol> 3，解离，充实和培养DRG神经元 - 第1部分<ol><li>地点锥形管与完整的DRG进入离心机，轻轻旋转（200 XG）2-3分钟。确认病种付费下沉到试管底部。如果没有，再旋转。 </li><li>用吸管，轻轻取出F12HS10，留下的DRG颗粒原状。加入2毫升1X钙，镁免费（CMF）的PBS，撞出病种付费的沉淀。再次旋转和删除CMF的PBS中，同时使DRG颗粒完好无损。重复，共洗涤三次这种洗涤步骤以去除在F12HS10胎牛血清，这会干扰胰蛋白酶消化在下一步骤。 </li><li>加入2mL温暖的胰蛋白酶15毫升管含2ml CMF的PBS中，按病种付费。在37℃水浴代替管10-15分钟来消化的DRG成解离细胞。 </li><li>在培养期间，使含有F12H +补充媒体（火腿的F12，10毫米的HEPES 100单位/ ml青霉素，0.1毫克/毫升链霉素，10毫微克/毫升NT3，10毫微克/毫升的NGF，200mM的L-谷氨酰胺和N 2）和温暖在37℃水浴。通常情况下，将10毫升的总媒体是足够的初级感觉神经元的4-5 35mm培养皿。也开始解冻层粘连蛋白-1涂布的盖玻片（步骤4）。 </li><li>与P1000吸管胰蛋白酶溶液轻轻磨碎病种付费和目视检查的完好无病种付费的。继续研磨直至完整的DRG通过眼睛不再可见和/或允许在37℃水浴中再胰蛋白酶孵育，直到完整的DRG解离。为了保护DRG神经元免受损伤，避免研磨过程中产生气泡，并限制胰酶消化&lt;30分钟。 </li><li> Centrifugê管含有游离病种付费，并在200 XG胰蛋白酶3-5分钟，形成的颗粒。如果需要的话，直到沉淀形成旋转更长的时间。 </li><li>小心吸胰蛋白酶，而不破坏球。加入2mL F12HS10的沉淀。轻轻磨碎病种付费使用P1000的枪头。 </li><li>转移15 mL锥形管的所有内容复制到100毫米培养皿。冲洗用8ml F12HS10的管，并将其转移到培养皿中，加入2ml在同一时间，总共10毫升。这个漂洗步骤有助于收集细胞，可能仍然附着到所述管的壁上。 </li><li>放置百毫米培养皿（10毫升F12HS10及解离的DRG细胞）成加湿37℃培养箱中培养3小时。 注:CO 2，不需要在孵化因为HEPES缓冲介质的pH在不存在的CO 2。在培养过程中，神经胶质细胞结合组织培养塑料和神经元的表面趋于松散坚持胶质床。 </li></ol> 4，涂层酸洗和烤盖玻片与层粘连蛋白-1 <ol><li>解冻在冰上冷冻层粘连蛋白-1的无菌分装。 </li><li>在无菌条件下，加入1毫升无菌1×PBS中的层粘连蛋白-1的50μl的等份。 </li><li>用分光光度计在280nm处得到的层粘连蛋白-1的吸光度值。使用下面的公式该值来确定的层粘连蛋白-1的浓度。 </li></ol> ABS 280 =（浓度毫克/毫升）*（蛋白的吸光系数） 消光系数为层粘连蛋白-1是0.86。 <ol start="4"><li>在无菌条件下，稀释的层粘连蛋白-1用1X PBS中，得到的浓度为1mg / ml和20毫克/毫升。使用这两种施加浓度的层粘连蛋白-1的实现层粘连蛋白-1孵育7,8,10后结合至盖玻片（30毫微克/厘米2和300毫微克/分别厘米2）的10倍的差异。 </li><li>在层流罩，用细镊子将一个酸洗和烤çoverslip入之一35毫米培养皿的底部。根据需要重复，以获得所需的菜的数量。 </li><li>若使盖玻片于UV光20-30分钟，以进一步确保灭菌。在此步骤期间保持盘盖关闭。 </li><li>添加400μl的制备层粘连蛋白-1的每个玻璃盖玻片。用枪头扩散层粘连蛋白1，确保盖玻片的全覆盖。表面张力将使层粘连蛋白1留在盖玻片和未​​涂布在培养皿上，这是需要的底部。 </li><li>放置盖子上盘和允许的层粘连蛋白-1温育2-3小时，在室温下（3小时是最佳的）。 </li><li>后层粘连蛋白-1孵育，冲洗用无菌技术与无菌PBS在层流罩盖玻片。删除解决方案在盖玻片，加入400微升PBS。等待5-10分钟。重复共3冲洗。 </li><li>最后一次冲洗后留下的PBS在盖玻片。冲洗时不要破坏表面张力，不允许盖玻片干。乐直到准备盖大道板块分离的神经元上coverlip。 </li></ol> 5，解离，充实和培养DRG神经元 - 第2部分<ol><li>孵育后，用10毫升无菌吸管轻轻漂洗含分离的DRG细胞的100mm培养皿的底部。操盘时〜45°角，拉〜7毫升F12HS10用吸管援助，然后轻轻驱逐7毫升到大约菜的三分之一。重复5倍，轻轻撞出神经元。 </li><li>旋转盘和重复漂洗程序，对于另外六个冲洗，上的菜的另一个三分之一。再次重复步骤剩余菜的三分之一。以这种方式，轻轻冲洗培养皿的整个底部与F12HS10除去神经元。 </li><li>使用相同的吸管，由100毫米的菜传输含F12HS10神经元至15锥形管。离心5-10分钟，以200g以沉淀细胞。 </li><li>删除F12HS10，留下沉淀不受打扰。悬浮颗粒用2毫升温F12H +补充剂。 </li><lI&gt;使用血球计数器，以确定细胞密度。 </li><li>稀必要与F12H +补充细胞以获得期望的镀浓度（120,000个细胞/ ml的盖玻片涂覆有1μg/ ml的层粘连蛋白-1和40,000个细胞/毫升的盖玻片上涂覆有20微克/毫升的层粘连蛋白-1 8,16）。 </li><li>从层粘连蛋白包被的盖玻片除去PBS中并立即将400μl的细胞上的盖玻片。精心调菜的潮湿，37℃细胞培养孵化器。允许温育至少1小时和最多3个小时，使神经元的粘附层粘连蛋白-1。 </li><li>在无菌条件下，轻轻地涌入35mm培养皿含有神经元与1.6 F12H +补充毫升。 请注意:这时，表面张力对盖玻片可破。神经元有足够的坚持层粘连蛋白1的盖玻片。 </li><li>回到细胞培养箱中孵育过夜。进行实验，如免疫第二天。 </li></ol> 6。我mmunocytochemistry <ol><li>之前，免疫细胞化学，在37℃水浴中温暖固定液（4％多聚甲醛，30％蔗糖2X PBS）中。 注:在固定的细胞中，用不同的试剂，如的netrin-1的Mn 2 +，音猬蛋白，脑信号蛋白，肝配蛋白和16,29-31。 </li><li>慢慢松开1毫升的2毫升F12H +补充从培养皿中。轻轻地加入1毫升温固定液。允许在室温下孵育10-15分钟。 </li><li>小心地取出固定液，加2毫升的PBS。应用解决方案，对菜的边缘，避免因应用程序的PBS固定细胞可能撞出。在室温下孵育5分钟。 </li><li>重复的PBS洗涤，共洗涤5次。不要让细胞在免疫细胞中的任何一步干燥。 </li><li>删除的PBS。申请1毫升封闭溶液（0.1％的Triton-X，1X PBS，5％正常山羊血清）。保温1小时，在室温下进行。或者，染色表面分子不要在封闭液8使用的Triton-X。 </li><li>稀释一抗堵到每个制造商的建议解决方案。用1:500的抗NCAM并激活β1整合素16。除去封闭液，加入​​1毫升的第一抗体溶液孵育1小时，在室温或过夜，在4℃。 </li><li>删除主要抗体溶液，加入2毫升的1X的PBS中。孵育在室温下5-10分钟。重复，共4 PBS洗涤。洗涤可以延长，如果需要的话。 </li><li>在封闭溶液1,000:稀释的二级抗体1。除去PBS，加入1 ml的二级抗体溶液孵育1小时，在室温下进行。 注:二级抗体是光敏感。保持溶液和细胞在黑暗中尽可能多地从该点向前。 </li><li>去除二级抗体溶液和冲洗5×用1×PBS，如以前那样。 </li><li>应用〜60-80微升Fluoromount的ğ到显微镜载玻片。用细镊子，轻轻抬起盖玻片出来的菜，下盖玻片成Fluoromount的G到显微镜载玻片。避免气泡产生。 </li><li>店内水平滑动，在4℃下在黑暗中。经过12-24小时，Fluormount G将先聚合和盖玻片将被牢固地附着在载玻片上。后该聚合步骤的图像的细胞是完整的。 </li></ol>

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作者什么都没有透露。

在这里，我们提出了详细的协议进行分离培养分离从鸡胚感觉神经元。这个过程产生强劲增长的神经元的体外 7,8,10,16富集群体。众多的细胞和分子生物学技术可以应用到这些培养的​​神经元中，包括免疫细胞化学，在此所说明。该协议是最近使用的定量评估免疫标记激活整合素在感官生长锥8,16力度。在这些先前的研究中，软件程序被用于准确地创建了生长锥的顶端20微?...

神经元是复杂的细胞传递信息的各种功能，包括感觉，视觉，运动运动，学习，记忆至关重要。从其他细胞类型的独特，神经延长臂状突起，称为轴突，以形成必要的神经公路进行通信。在位于轴突生长的秘诀开发专门车厢，称为生长锥，浏览外线索的音乐会导致轴突到相应的目的地。背后生长锥的导航复杂的分子机制尚不完全清楚。为了更好地理解这些机制，研究人员已经用细胞培养模型，研究了定义，并简化在体外环境中的神经元。在文化学习1神经元，导致了我们的神经元细胞生物学的理解，包括显著进展:神经细胞的分化2，细胞骨架动力学，细胞内吞作用和贩运，枝晶规3,4，轴突再生5，和临床病症，如神经病6。此外，培养的神经元是高度适合于范围广泛的研究的技术，包括免疫细胞化学，细胞表面的共免疫沉淀，Western印迹，转染，RNA干扰，和实时成像等的生长锥运动缩时分析。因此，培养初级神经元是一种有效的方法来阐明神经元的细胞生物学的许多方面。 细胞培养模型提供与调查员对环境条件和变量详细的控制。例如，在其上的神经元被镀（并且在生长）的基质可以容易地操纵。这里，我们提供了用于产生两个不同的基质，一种具有低的层粘连蛋白-1的浓度和其它具有饱和的层粘连蛋白-1的浓度的详细说明。出人意料的是，不同浓度的相同的分子可具有显着影响对神经元的内部状态，以及它们的细胞表面成分。例如，整合的cAMP和表面水平的细胞内水平是在神经细胞铺于两个基质7,8显著不同。另外的研究表明，其它分子，包括纤连蛋白和硫酸软骨素蛋白多糖，影响细胞表面分子的表达和神经元运动7-11。此外，可溶性分子如神经营养因子和neurotropins也影响细胞膜的组成和神经运动12-16并且可以在细胞培养模型中很容易地和准确地操作。 在这里，我们描述的方法来分离和培养解离来自鸡胚的感觉神经元。这个过程已经被用来制造在神经生物学显著突破，包括轴突生长5,7,8,10,11,16-21和从设计到分离神经节细胞22中的程序进行了修改。有几个优点这种方法。首先，鸡背根神经节（DRG）开发的许多功能都得到很好的特点，包括出生，轴突延长的时间框架和蛋白质表达谱2,23-28，从而提供了赖以建立翔实的体外实验的指导性依据。第二，解离的神经元培养物使研究者能够更直接地学习的神经元相比，使用含有两个游离的神经元和非神经元细胞完好的DRG外植体（其包含神经元和非神经元细胞）和/或混合培养物的替代方法。第三，这里描述的方法很简单，价格低廉，适合于大学生。因此，这种技术可以用于研究以及用于教学目的。此外，该协议的微小变化应该让神经元并非来自病种付费等速度快，产量高的净化。例如，这个过程可以被修改以提供neuronally ENR从其它组织iched培养如胚胎前脑或脊髓。 免疫细胞化学的协议已经被优化用于这些分离的神经元培养物和这里进行了详细的说明。提供了一种用于对神经细胞粘附分子（NCAM）和β1整合素双重免疫细胞的步骤。从这些免疫细胞化学方法产生的数据已被用于检查空间构图和几个分子的强度在培养的神经元8,16。

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile small culture dishes (35mm)</td>
			<td style="width:176px;">Corning</td>
			<td style="width:135px;">430165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large culture dishes (100mm)</td>
			<td style="width:176px;">Falcon</td>
			<td style="width:135px;">353003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large petri dishes (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">89000-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass petri dish (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">D108962&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">11251-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">standard forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">1100-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">coverslips&#160; (22x22)</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:135px;">12 518 105K</td>
			<td>0.13-0.17 mm thickness is optimal for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">porcelin coverslip holder</td>
			<td style="width:176px;">Thomas scientific</td>
			<td style="width:135px;">8542E40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine serum</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">17502-048</td>
			<td>use 50ml aliquots per 500 mls of Ham&#39;s F12 media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCL</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">VW3204-1</td>
			<td>use at 2M, can be used twice before discarding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9888</td>
			<td>use 5M NaCl solution for laminin-1 stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">laminin-1</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">23017-015</td>
			<td>Add 72ul of sterile 5M NaCl and aliquot into 50ul</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical tube</td>
			<td style="width:176px;">cell treat</td>
			<td style="width:135px;">229411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS CMF 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14200</td>
			<td>used at 1X, dilute with sterile millipore filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14080</td>
			<td>used at 1X, dilute with sterile milliporee filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ham&#39;s F12</td>
			<td style="width:176px;">Lonza</td>
			<td style="width:135px;">12-615F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">10438</td>
			<td>use in F12HS20 at 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">H3375</td>
			<td>make sterile 1M solution in distilled water, dilute in F12 media to obtain final concentration of 10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin streptomyocin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P0781</td>
			<td>use 5mls in 500mls of F12 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NT3</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">GF031</td>
			<td>Add 1ml of sterile millipore water, aliquot under sterile conditions, store at -20 0C, use at final concentration of 10ng/ml in F12H media, keep in -20 C&#160; non-defrosting freezer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">17502048</td>
			<td>&#160;aliquot under sterile conditions,stored at -20 0C,&#160; use 100ul per 10ml of F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NGF</td>
			<td style="width:176px;">RnD systems</td>
			<td style="width:135px;">256-GF</td>
			<td>Add 1ml of sterile 1X PBS with 0.1% BSA, aliquot under sterile conditions, store at -20 0C, usd at final concentration of 10 ng/ml in F12H media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">l-glutamine</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">G7513</td>
			<td>aliquot under sterile conditions, store at -20 0C, use at final concentration of 200mM in F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T4049</td>
			<td>aliquot under sterile conditions, store at -20 0C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">15260</td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">Other items needed:&#160; general dissection instruments, including&#160; glass pasteur pipettes, fertile white leghorn chicken eggs, check egg incubator (humidified, 37 degrees C), cell incubation chamber (humidified, 37&#160; degrees C), laminar flow hood, binocular stereovision dissecting scope</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immunocytochemistry Reagents Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9378</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X 100</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluoromount G</td>
			<td style="width:176px;">Southern Biotech</td>
			<td style="width:135px;">0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">PCN500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microscope slides</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">16004-430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibody against NCAM</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">AB5032</td>
			<td>polyclonal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Antibody against activated beta 1 ingegrin</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">MAB19294</td>
			<td>monoclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Seconday Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-mouse IgG Alexa 488</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-rabbit IgG Alexa 548</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11036</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of dissociated sensory neurons from chick embryos

神经元是多层面的细胞中携带信息的各种功能，包括感觉，运动运动，学习和记忆至关重要。 体内研究神经元可以是具有挑战性的，因为它们的复杂性，其变化的动态环境，和技术的限制。由于这些原因， 在体外研究神经元可以证明是有益的解开神经元的复杂的奥秘。的细胞培养模型中良好定义的性质提供了对环境条件和变量的具体控制。在这里，我们介绍了如何分离，分解，并从鸡胚培养初级神经元。这种技术是快速，廉价，并且产生强劲增长的感觉神经元。该过程持续产生培养物被高度富集的神经元，并具有很少的非神经元细胞（小于5％）。原代神经元没有很好地附着在未经处理的玻璃或组织培养塑料，因此详细的程序来创建两个迪斯丁CT，定义良好的含有层粘连蛋白基质为神经镀进行说明。培养的神经元是高度适合于多种细胞和分子生物学技术，包括免疫共沉淀，活细胞的想象，RNA干扰，和免疫细胞化学实验。关于这些培养的​​神经元的双免疫程序进行了优化，并在此描述。

这里所描述的协议使研究者培养解离胚胎感觉神经元具有很少（ 例如 ，&lt;5％）非神经元细胞7,8,10,16的富集群体。许多病种付费可从腰骶部，胸椎和颈椎区域获取。根据研究者的需要，从这些不同的解剖区域的DRG可以容易地分离。例如， 图1示出了鸡胚通过夹层与腰椎的DRG不同阶段的图像在图1C和图1D和图1E和1F胸椎的DR...

Watch this Scientific Journal Video about Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos at JoVE.com

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

细胞培养模型提供了环境条件的详细控制，从而提供一个强大的平台，以阐明神经元细胞生物学的许多方面。我们描述了一种快速，廉价和可靠的方法来从鸡胚分离，解离和培养的感觉神经元。还提供基质制剂和免疫细胞化学的细节。

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-culture-dissociated-sensory-neurons-from-chick

Research

JoVE Journal

Neuroscience

14.2K 次观看.  Assumption College. 细胞培养模型提供了环境条件的详细控制，从而提供一个强大的平台，以阐明神经元细胞生物学的许多方面。我们描述了一种快速，廉价和可靠的方法来从鸡胚分离，解离和培养的感觉神经元。还提供基质制剂和免疫细胞化学的细节。 

视频: Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

初级运动和感觉神经元上的静电聚- L -乳酸纳米纤维支架中定义的媒体文化

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

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 of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

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

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

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

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

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

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

分离和游离感觉神经元的文化活动从鸡胚

摘要

探索更多视频

此视频中的章节

从胚胎脊髓连合神经的解剖和文化

初级运动和感觉神经元上的静电聚- L -乳酸纳米纤维支架中定义的媒体文化

从产前小鼠海马神经元的分离和培养

隔离的感觉神经元中

从胚胎前脑内皮细胞的分离和培养

嗅觉受体神经元在全山免疫标记

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

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斑马鱼

分离和游离感觉神经元的文化活动从鸡胚

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

初级运动和感觉神经元上的静电聚- L -乳酸纳米纤维支架中定义的媒体文化

从产前小鼠海马神经元的分离和培养

隔离的感觉神经元中

从胚胎前脑内皮细胞的分离和培养

嗅觉受体神经元在全山免疫标记

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

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斑马鱼

从胚胎脊髓连合神经的解剖和文化