This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

注：该动物保持并遵守处理与制度的指导方针，所有动物的程序批准了帝国学院的动物福利和伦理审查机构（AWERB）以及内政部和哈佛大学的机构动物护理和使用委员会（IACUC）符合联邦和州法规。 1，试剂和设备安装<ol><li>解冻，分装和存储1日-80ºC毫克/毫升层粘连蛋白的解决方案。溶解2微升在1ml DMEM / F12，导致在1-2微克/厘米2的涂布的浓度。 注：解冻层粘连慢慢在冰上并溶解在冷的DMEM / F12，并立即将其添加到板/盖玻片。 </li><li>稀释75毫升0.01％聚-L-鸟氨酸425毫升的PBS，并储存于4ºC。 注：工作液的保质期长达一个月。 </li><li>溶解25％（重量/体积）BSA的PBS，pH 7.4中，并储存在4ºC长达一个咋AR。 </li><li>制备50％FBS（在HBSS中）的失活的介质。 </li><li> （+） - - 葡萄糖（重量/体积），0.25％的BSA（重量/体积），以通过添加50U / ml的青霉素和链霉素，1×N2添加剂，5％（体积/体积）胎牛血清，0.36％D制备的完全培养基DMEM / F12，并储存在4ºC。 注：保质期是10天。 </li><li>在沸腾的70％（体积/体积）乙醇为至少30分钟的地方盖玻片以除去油脂和高压釜任何痕迹。 </li><li>在24孔板的地方盖玻片并加入500μl聚-L-鸟氨酸溶液到每个孔中。孵育所述组织培养罩下1小时，在室温下，然后，洗3次，用500μl水。加入500μl层粘连蛋白的解决方案，以每块板和孵化O / N在湿润的组织培养孵化器在37ºC。 注意：虽然没有洗涤是必要的层粘连蛋白处理后，洗涤该聚-L-鸟氨酸小于三倍导致细胞的死亡培养后24小时。 </li><li>擦亮玻璃吸管超过天然气或火炬f显示提示跛脚。 注意：在此步骤之后的尖端的直径应为约一半正常大小的。 </li><li>削减1.5 ml离心管的盖子，用剪刀蒸压釜和。 </li></ol> 2.解剖胚胎中脑腹侧的<ol><li> Narcotize定时怀孕（E12.5）的CO 2吸入水坝，然后由颈椎脱位安乐死。 </li><li>喷用70％乙醇的腹部，使腹部切口，直到子宫囊都暴露出来。使用镊子削减阴道附件，删除子宫。放置在冰冷的HBSS子宫，在100毫米培养皿中。 </li><li>除去从子宫和羊膜囊胚胎，使用镊子（ 图1C）。地方胚胎在新鲜的冰冷的HBSS。 注： 图1D的矩形表示胚胎中脑腹侧的位置。 </li><li>将解剖显微镜（10倍放大倍率）在胚胎和解剖脑拱通过切割大脑在峡部和中脑-间脑边界区域，使用bioscissor和镊子（ 图1E，请参考图1A，用于切口线）。 </li><li>后取出整个脑，做了脑内侧背（ 图1F）削减。 </li><li>除去脑膜，使用两个镊子（ 图1H）。 注：关键步骤：此步骤是为最佳的神经元的产量和存活至关重要。由于培养条件是最佳的脑细胞中，大部分从周围组织中的细胞死电镀后，并从该组织中的细胞凋亡信号可能损坏培养物的完整性。 </li><li>拼合在培养皿中脑组织，观察蝴蝶造型和削减大约各占一半机翼，用消毒的剃须刀片（ 图1H，I）。 注： 图1J描绘了孤立的腹侧中脑。 </li><li>转让一块腹脑入冰冷的HBSS在一个15毫升的锥形管，然后继续下一个胚胎。 注意：步骤2.1-2.8不应超过1小时。保持胚胎超出在冰上这个时间帧显著降低细胞活力。 </li></ol> 3.解离腹侧中脑细胞<ol><li>传送下一个层流罩腹侧中脑的部分。移除HBSS和加入1 ml预热（37℃）0.05％胰蛋白酶-EDTA入锥形管（体积足够容纳12个腹侧中脑）。孵育所述组织在37ºC5-10分钟。 </li><li>除去胰蛋白酶-EDTA罩下和加入1毫升去活化介质（血清将停用胰蛋白酶）来组织。 </li><li>取出失活介质和洗涤在1ml完全培养基中的组织的两倍。 注：避免除去从管的所有介质和吸取的组织，以防止废弃离解的细胞。 </li><li>加入1ml完全培养基和三叔尿酸盐用火抛光的玻璃吸管的组织。避免形成气泡，并继续直到捣碎单细胞悬浮液来实现的。 注：通常情况下，8-10通过限制提示所需的完整的解离。 </li><li>离心细胞，在400×g离心5分钟，在室温并取出介质。 </li><li>重新暂停通过缓慢吹吸在1ml完全培养基中的沉淀和向下多达四次。 </li></ol> 4.电镀中脑腹侧细胞<ol><li>混合10微升细胞悬浮液与90微升锥虫蓝溶液，并用血细胞计数器计数细胞数。 注：细胞的生存力，也可以在此阶段进行检查。 </li><li>放置无菌微量离心管帽在100mM培养皿转移聚-L-鸟氨酸/层粘连蛋白包被的盖玻片，使用镊子，一次一个上盖帽的顶部。 注意：不要洗盖玻片，避免干燥的层粘连蛋白，因为这可能会导致uneveñ文化和减少细胞的生存能力。 </li><li>加入完全培养基调节音量，以每1微升1500细胞并在每个盖玻片的顶部添加细胞悬浮液的100微升（总共150,000个细胞的）（体积为最佳，以覆盖盖玻片的表面而不溢出）。关闭培养皿孵育它们1小时，在湿润的组织培养箱（37℃，5％的CO 2）。 注：关键步骤：此步骤是必需的用于增强细胞的附着和生活力并增加每个胚胎盖玻片的数量。或者，所述细胞可以直接转移到孔中（含有盖玻片），但是单元的数目应增加（而不是150000），以350000每孔。换句话说，使用这种方法，8-10盖玻片能够获得，而直接转移产生，因为细胞，其附着到所述板（旁或盖玻片下）为约3-4盖玻片。平均viabiliTY培养物，使用该方法是在90％左右。 </li></ol> 5.文化成长和维护<ol><li> 1小时温育后，小心地转移盖玻片包括介质到24孔板，含有400微升预热（37℃）的完全培养基（总体积：500微升）。孵育细胞O / N为37ºC。 </li><li>内孔中温育24小时后，轻轻地加入500微升完全培养基到每个孔中。 注：第几个小时为多巴胺能神经元的存活和存活细胞的数目不显著超过该点改变的最关键的阶段。 </li><li>不附加媒体添加到培养的头两个星期或直至介质变为黄色。如果对于超过两周或介质颜色变为黄色，介质的交换一半（500微升）与新鲜的完全预热介质（典型地每两个星期）培养。 注：内多巴胺能神经元培养将生存在这些条件下6周以上。 </li></ol>

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No conflict of interest declared.

在中脑多巴胺能神经元是多巴胺在中枢神经系统中的主要来源。它们被分成三组，黑质致密部（黑质致密部），腹侧被盖区（VTA）和retrorubral字段（RRF）10，11。在黑质致密部和VTA的神经元产生重大多巴胺能通路，mesocortical，脑边缘和黑质纹状体，涉及的功能，如情感，动机和运动行为的控制。在黑质致密部和黑质纹状体系统的功能性破坏的神经元死亡是第二个最显着的神经变性疾病，帕?...

多巴胺能神经元从黑质损耗致密部导致帕金森氏病（PD），所述第二最常见的神经变性疾病的基本运动症状。这种脑的神经元群的灭亡的根本原因尚不清楚。研究负责开发和调制神经生理特性和mesDA神经元，几个细胞培养和动物模型系统的生存已使用的生化途径。永生化细胞系，包括大鼠多巴胺能细胞系1RB 3 AN 27（N27），所述的SH-SY5Y人的多巴胺能神经母细胞瘤细 ​​胞系，小鼠的多巴胺能杂交细胞系MN9D和人脑LUHMES细胞已被用于生化和有限的机理研究1 -5。对于mesDA神经元的具体损失的研究，几种神经毒素为基础和遗传模型已开发6-8。原发性脑腹侧文化，提供一个不可缺少的工具用于研究多巴胺能神经元的神经元和突触的属性和参与这一常见的疾病的发病机制中的通路。 这里我们提出了一个详细的协议，用于中脑多巴胺能神经元的分离，它含有导致更高的生存能力和每个胚胎盖玻片的增加的产量的修改。使用预成熟E12.5鼠脑（E14.5大鼠）提高了生存能力。在这个年龄段的神经元还没有开发轴突的是，它在清扫树叶细胞的完整，减少应力，从而提高显著生存能力。此外，小心解​​剖腹侧中脑，如在该协议的第2节描述的，进一步提高的生存能力。增加每个胚胎盖玻片的数量，另一种电镀方法，提出在该协议的第4条。这导致每个胚胎高达10盖玻片的收率相比，下4盖玻片标准电镀条件从而减少每个实验动物的数量。 在轴突和树突的文化展品生长的神经元，形成突触联系，揭示了神经元和突触的标记使这些文化适合活细胞成像，免疫细胞化学和电生理研究的存在。此外，使用的神经元培养物的促进基因和药理学操纵。 体外 2天的轴突生长允许发育研究。此外，培养物的长期存活（最多6周），使它们适合于这些神经元的速度慢，进行性变性的研究。

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:364px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified Eagle medium nutrient mixture F-12</td>
			<td>Invitrogen</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td>Invitrogen</td>
			<td>24020-117)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum, heat-inactivated (FBS)</td>
			<td>Invitrogen</td>
			<td>16140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td>17502-048)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose solution (45% (wt/vol) in water</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin BSA</td>
			<td>Sigma</td>
			<td>A9430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% (wt/vol)</td>
			<td>Invitrogen</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA) cell culture tested</td>
			<td>Sigma&#160;</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Tyrosine hydroxylase (Th) antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td>P60101-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-ornithine, 0.01% solution</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td>
			<td>Millipore</td>
			<td>MAB3418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Synapsin-1 antibody</td>
			<td>Millipore</td>
			<td>AB1543P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-mouse IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue solution (0.4% (wt/vol)</td>
			<td>Biowhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted phase contrast microscope</td>
			<td>Carl Zeiss</td>
			<td>Axiovert 40 C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>May-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover Slip Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11251-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two Dumont #45 Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11245-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blade Holder/Breaker Flat Grip - 11cm</td>
			<td>Fine Scientific Tools</td>
			<td>10052-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Iris Scissors - Straight 11.5cm</td>
			<td>Fine Scientific Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber optic halogen illuminator</td>
			<td>Nikon</td>
			<td>MKII</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Proscitech</td>
			<td>SVZ2NIOU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m sterile filter units</td>
			<td>Nalgene</td>
			<td>NL-CE-156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100x20mm Petri dishes</td>
			<td>BD Biosciences</td>
			<td>351005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Cover Slip #1 Thickness German Glass 12mm</td>
			<td>Bellco Glass</td>
			<td>1943-10012&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

免疫组织化学对酪氨酸羟化酶（TH）表明，该细胞在培养中的0.5-1％之间的多巴胺。电镀后的2小时的神经元突起出现并通过第一天，轴突和树突是可区分（ 图2），使用酪氨酸羟化酶（TH）和微管相关蛋白2（MAP2）抗体（ 图3）。神经元存活超过六个星期，并显示大量的产物。在培养的神经元 - 神经胶质细胞比直接相关的血清的培养基中的浓度，如前所示。同时消除FBS从培?...

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

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

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 ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Research

JoVE Journal

Neuroscience

13.8K 次观看.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’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.

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

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

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 ...

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 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 ...

Primary Culture of Mouse Dopaminergic Neurons

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

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

小鼠多巴胺能神经元的原代培养

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

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. ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...