我们感谢丹尼斯FLIPO用于图像处理和分析意见，戴维W在银鱼的实验室生成的Gata4p [5KB]-GFP的小鼠品系。研究在实验室皮隆是由CIHR，NSERC，FRQS和FRQNT资助。

伦理声明在老鼠的实验进行了以下的在医学研究中使用动物的照顾和处理动物护理指引加拿大议会。涉及动物的操纵协议已获魁北克蒙特利尔大学的伦理委员会（科米特Institutionnel去保护DES ANIMAUX;编号0512-R3-650-0513）。 1。胶原蛋白凝胶的制备在无菌的方式工作，在组织培养罩。 <ol><li>准备完整的5倍DMEM培养基（Eagle的修改必需培养基），包括标准的抗生素。溶解3.37克DMEM粉末和0.925克的NaHCO 3在20毫升水中。穿过一个0.22微米的过滤器进行消毒。添加2.5无菌100X青霉素/链霉素和25毫升无菌热灭活胎牛血清的溶液中。贮存于4℃。 </li><li>冰，混合800微升的I型胶原溶液（3.77 mg / ml的0.02N的乙酸，过滤灭菌的），600微升的完全DMEM培养液5倍和17微升1N的NaOH。用无菌水稀释至3毫升的最终体积。包括在组合有关的生长因子。我们通常使用的GDNF在10毫微克/毫升，以刺激肠道NCC迁移。 </li><li>存款约480μl，以单行的在24孔板中每个孔中。避免气泡。其余行可用于测试的其他生长因子对细胞迁移的影响。 </li><li>让胶原蛋白在37℃下聚合至少1小时，在无菌恒温箱</li></ol> 2。 16动物的解剖<ol><li>成立交配和检查阴道塞第二天早晨。一天E0.5是中午阴道塞被发现的当天，隔离女性和等待12天，直到E12.5。 </li><li>麻醉孕鼠与异氟醚和二氧化碳吸入安乐死。 </li><li>喷鼠标，用70％的乙醇。提起腹部s亲属，并与解剖剪刀打开腹部腔。 </li><li>取出子宫到玻璃培养皿中充满了冰冷的PBS（磷酸盐缓冲盐水）。切子宫横向个人蜕膜肿胀之间的隔离每个胚胎着床部位。 </li><li>每个植入位点在另一个玻璃培养皿充满冰冷的PBS分别工作。在解剖显微镜下，用细镊子取出子宫肌肉层。 </li><li>打开卵黄囊和羊膜揭示胚胎。切断血管加盟胚胎的胎盘/卵黄囊的时候，因为他们是交织在一起的发展肠子照顾。 </li><li>断绝胚胎的头颈部。 </li><li>插入一个封闭镊子在腹腔胚胎的空腔，就在暗红色色肝上，并让钳子打开的本身（停止施加压力），以使一个横向开口在腹部空腔中。抠开镊子下来，对胚胎完全打开腹部的后端。 </li><li>抢肝背后的结缔组织和拉的胆量了腹部，注意不要打破肠（大肠连接到肛门）。 </li><li>切断结肠，从胚胎的其余部分释放的胆量。切割可以在沿着结肠的任何位置进行。保留尾部后。 </li><li>梳理出从盲肠结缔组织，肠子然后休息。要小心，不要伤了肠子，而这样做。 </li><li>切掉肝和胃（在小肠的喙末端），以及中肾和生殖嵴，如果一些都存在。 </li><li>隔离小肠。再次，要小心不要夹到肠。从现在起，肠道组织的rosrrocaudal方向可以使用锋利的曲率目前在喙末端进行跟踪。留在小肠的PBS中于室温下尽可能短的时间尽可能烯前床上用品（见步骤3.3）。 </li><li>最后，记录尾部体节的每个胚胎的数目准确地确定胚胎发育的阶段。 </li></ol> 3。胚胎肠切片<ol><li>在继续进行胚胎解剖，熔体1.5克琼脂糖于100ml PBS（磷酸盐缓冲盐水），并保持在50℃。 </li><li>倒入琼脂糖融化成一个嵌入模具（ 例如闭2 ml离心管，已被切割纵向切除约1/4的管壁）。让琼脂糖冷却至约42-45℃，它应该是只稍微温暖的触感。 </li><li>就在琼脂糖固化之前嵌入胚胎肠（这可以用钳子提示进行评估，并会发生大约36-38℃）。保持由所述折叠喙端与钳肠道，通过沿着模具的长度琼脂糖拉得非常缓慢。这有助于保持肠道直而琼脂糖集。放开T只要它开始反抗移动的问题。跟踪肠道的喙，尾方向。 </li><li>放于冰箱中2-3分钟的模具，以确保琼脂糖完全凝固。 </li><li>取出琼脂糖从模具中（通过向外滑动打开的Eppendorf管中）。用刀片，取出多余的琼脂糖两端，使切口垂直于肠。 </li><li>胶水肠/延髓结束琼脂糖挡了下来与振动叶片切片机的金属舞台。修剪对肠/琼脂糖块两侧的过量的琼脂糖。 </li><li>安装在上述振动切片机室中的金属的阶段。如果有必要，调整阶段的角度，使肠道是尽可能垂直（因而垂直于叶片）。盖在冰冷的PBS试样。使切片机刀片向下到几毫米下的缓冲表面。 </li><li>使200微米vibratome横向切口尾最小肠，确保该电子ACH琼脂糖片包含一个完整的肠道部分。 </li></ol> 4。肠道外植体的培养<ol><li>轻轻的存放新鲜切肠/琼脂糖片平放在胶原凝胶用钳子，把一片鸡犬中间好。 </li><li>孵育3天37℃，在潮湿的5％CO 2的气氛中，以允许NCC的迁移出来的外植体。 </li><li>取小肠/琼脂糖片落胶原凝胶轻轻地用钳子。小心不要损坏凝胶下方。 </li><li>避免在为了随后的温育和洗涤步骤直接触摸胶原凝胶，以确保细胞迁移模式不受影响。修正了每孔1小时，在室温下500微升4％PFA（多聚甲醛）（在PBS）中。 </li><li>更换固定液，用500μL的DAPI（4&#39;，6 - 二脒基-2 - 苯基吲哚）溶液（5μg/ ml的PBS中），每孔，孵育10分钟，在室温下进行。 </li><li>每次洗好3倍用500μlPBS中5分钟。 </li><li>拍摄嵌入在胶原凝胶内各孔的荧光细胞（GFP和DAPI通道）。 </li></ol> 5。图像分析我们广泛利用ImageJ的17来处理和量化外植体培养后生成的图像。 <ol><li>首先，成像微米幻灯片在相同的放大倍数为荧光细胞的照片。测量以像素为单位一微米（用直线工具）的长度。 </li><li>设置比例（分析/设置刻度;像素/微米输入号码）。 </li><li>对于每个GFP荧光照片，图像格式改变为8位灰度（Image/Type/8-bit）。 </li><li>调整信号（图像/调整/亮度/对比度）的强度。 </li><li>减去背景噪音，如果有必要（工艺/减去背景;调整滚球半径）。 </li><li>设定阈值以突出细胞和细胞团块（图像/调整/阈值），则Ñ​​应用分水岭分割团块（工艺/二进制/流域）。 </li><li>分析粒子指定感兴趣区域（ROI）的区域，并生成细胞数量的统计数据（分析/分析颗粒;集大小最小排除剩余的像素）。 </li><li>设置测量选项包括费雷特直径（分析/设置测量）。 </li><li>集团的投资回报，并测量作为一个整体来确定的Feret直径，指示细胞扩散（分析/工具/投资回报率经理/更多/ OR，则投资回报率经理/添加和ROI经理/测量）。 </li></ol>

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作者什么都没有透露。

我们展示了如何我们的体外外植体培养技术可以用来精确地量化肠溶NCC迁移潜力的GDNF的存在。这种精确的定量是通过使用200微米厚的vibratome肠组织切片而不是大块的近似大小，如先前所描述11,12,15大大便利。的确，这使得我们能与细胞中一种高度可重现设定合理数量的工作。值得注意的是，荧光肠溶NCC的小肠从该外植体条带被切断的尾最区域内的均匀分布还允许从一个单一的肠道?...

神经嵴细胞（NCC）是胚胎发育过程中形成了许多衍生工具的瞬态细胞类型特有的脊椎动物。该细胞群出现在神经板，相邻的非神经外胚层1的边界。在神经胚形成，为神经板的地方NCC沿形成神经管背侧边缘弯曲。 NCC再经过上皮 - 间质转化，分离和迁移远离神经管。 NCC殖各个胚胎结构，包括消化道那里形成整个肠神经系统（ENS），神经节的互连网络中嵌入肠壁。由于最近检讨2,3，许多基因都参与了这个复杂结构的发展。 大多数ENS的是从迷走神经管（ 即约准后脑/脊髓边界）一个小水池NCC原始的派生4。这些神经祖细胞到达前肠围绕胚胎一天（五）9.0小鼠，然后尾端迁移肠道间质内，直到约e15.0拓殖整个胚胎肠。结肠的神经祖细胞的未成年子集也由骶NCC，其侵入后肠道的相反方向到盲肠4提供。既迷走神经和骶NCC需要多次迁移，增殖，存活和分化，促进线索，以确保完全形成ENS的。在这方面，动物模型 - 尤其是转基因小鼠 - 是有助的在几个必要的细胞外配体的鉴定：GDNF（神经胶质细胞源性神经营养因子），内皮素-3，神经营养素-3，骨形态发生蛋白（骨形态发生蛋白），netrin诱导，以及声波和印度刺猬（Shh和Ihh信号）5-10。其中，GDNF通过酪氨酸激酶跨膜受体RET（转染过程中整理的）信号被确认为日Ë最关键的途径的吸引力和NCC的指导，并在胚胎肠道。 GDNF是由肠道间质细胞分泌，并形成了一个时空方式控制rosrrocaudal梯度是直接chemoattractive到肠道NCC，其表达RET 11,12。 除其他功能外，ENS通过与肠壁平滑肌相互作用调节消化道内运动。在肠道导致先天性巨结肠的末端区域神经节的缺席：受影响区段的强直性收缩导致堵塞，消化材料的上游积累和肠道和腹部的大规模扩张。先天性巨结肠症发生大约在5,000一只活产婴儿。肠道NCC的rostro-尾迁移模式被认为是主要的促进因素先天性巨结肠症的病因。从迁移NCC和b的最后部分的源结肠，最远owel被殖民统治，是最容易受到ENS中形成的缺陷。按照肠道NCC迁移的关键作用，GDNF / Ret信号的干扰是先天性巨结肠13的主要已知的遗传原因。 为了更好地研究华北克拉通和ENS的发展，我们产生了转基因小鼠线-命名Gata4p [5KB]-GFP 14 -在这种迁徙NCC都贴有绿色荧光蛋白（GFP）。我们接下来完善的体外细胞迁移测定中，由其它组11,12,15适于从发表的作品，即现在允许在各种生长因子，如GDNF的存在肠溶NCC迁移潜在精确定量。

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

神经嵴细胞（NCC）是一个短暂的和多能细胞群源于背神经管和广泛地在整个发展脊椎动物胚胎迁移。除了提供周边神经胶质细胞和神经元，NCC生成的黑素细胞，以及大部分的颅面骨骼。 NCC迁移和分化是由它们的轴向原点沿神经管和他们接触到不同的区域外线索的组合控制。细胞外配体的这种贡献是特别明显的肠神经系统（ENS），神经节的一个复杂的相互连接的网络，本地控制（其中包括）肠道肌肉运动和肠蠕动的形成过程中。在吻端到尾鳍时尚 - - 准肠道的整个长度大多数ENS是从华北克拉通，为了殖民承接了漫长的旅程一个小的初始池派生。其中已知的多条信号通路影响肠道定植NCC，GDNF / Ret信号被认为是最重要的。事实上，RET配体GDNF的时空方式控制分泌肠道间质，主要是负责吸引和RET表达肠NCC指导，并在胚胎肠道。在这里，我们描述的体外细胞迁移测定，利用转基因小鼠系具有荧光标记的NCC，这使得在各种生长因子，包括GDNF的存在肠溶NCC迁移潜在精确定量。

下面的结果是代表什么可以与这里描述的（ 图1）的技术来获得的。使用生长因子（ 即 GDNF）刺激的表达GFP的肠溶NCC出肠道外植体并进入胶原凝胶（ 图2）的迁移。虽然一些小区出来的外植体在无生长因子，这些大多不是GFP标记和表示被动条目。有必要从胶原凝胶取出肠切片，以便记录的结果，因为这个组织仍然是人口稠密通过荧光细胞和本来隐藏的细胞趴在胶原?...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

我们提出了一个先体外后体内的细胞迁移测定法，允许在各种生长因子的存在下肠道神经嵴细胞迁移的潜在精确定量。

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K 次观看.  UQAM. 我们提出了一个先体外后体内的细胞迁移测定法，允许在各种生长因子的存在下肠道神经嵴细胞迁移的潜在精确定量。 

视频: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

跨物种移植神经嵴迁移和分化的分析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.