这项工作是由卫生科学中西部大学基金研究促进格兰特授予英皇酒店与研究中西部大学办公室和赞助节目校内格兰特授予KJL支持。

该协议符合AVMA动物福利原则实验动物的照顾和使用，已被批准的机构动物护理和使用委员会（IACUC）在中西部的大学。 1.准备麻醉，设备，及媒体<ol><li>脱氯约1.5升使用一个商业脱氯剂（可在宠物商店），或者通过让水静置24小时自来水。获得3 1升的烧杯中，并填写每用500毫升水脱氯。标签之一任何操作之前，拿着鱼和一个用于恢复。到第三烧杯中，加入三卡因甲磺酸盐的100微克/毫升;这将成为其中的鱼类会被麻醉的烧杯中。 </li><li>填充浅盘用冰冷却，温热的培养基（RPMI 1640,10％FBS（胎牛血清），50微克/毫升庆大霉素，100μg/ ml的卡那霉素，25毫米的HEPES）至RT，并收集干净珠宝商的镊子。干净的镊子由音麦唱在70％的乙醇的前端，并穿过本生灯，允许乙醇以烧掉。 </li><li>决定是否将被放大，而不执行的程序，与商业，台式照明放大镜2 - 10X放大倍率，或解剖显微镜（完全依赖于个人偏好）。 </li></ol> 2.建立植文化<ol><li>捉在保持烧杯鱼和地点的所需数目。传输单个鱼麻醉烧杯中;最好是单独麻醉鱼。 </li><li>监测麻醉通过观察鱼的游泳和鳃运动的影响。随着麻醉的进展，刺运动减慢，鱼会游泳不正常，经常倒挂;考虑鱼只要鳃运动停止麻醉。不要让鱼更长时间的麻醉洗澡，因为这可能会导致鱼的死亡。 </li><li>从麻醉烧杯并转移除去鱼冰，水平放置鱼面临的手拿着镊子的尾巴。如果经历了该技术，如果多个鱼将被用于制造植体，考虑在此时将所述下一个鱼在麻醉烧杯中。 </li><li>要去除鳞屑，位置镊子平行与尖鱼的规模优势。踩下镊子的平侧面略成鱼的侧，以使刻度从鱼的身体提升。把握规模镊子，勇气，和地点（无反转）在组织培养皿。重复该过程，除去一个最大值，从一个大的成人（6从各侧）12秤，避免了鳃区域，侧线，和翅片。 </li><li>将鱼放入烧杯中恢复和监控，以确保它从麻醉的效果恢复。此时，鱼可以被转移回单个罐。让鱼恢复了至少21天，以便完整规模的再生。 </li><li>根据实验设计，将高达塑料35mm组织培养处理的培养皿20天平或4 - 每个玻璃底菜6尺度。让秤坚持的菜才轻轻添加媒体以免分离从盘刻度。 注意：使用的经验，前介质被添加到第一盘的多个鱼都可以加工。 </li><li>添加1.2毫升完整的媒体（见1.2以上）到每个35mm培养皿。 </li><li>在5％CO 2孵育优选外植体培养在28℃的时间有或没有额外的处理所需的时间。另外，孵育在加热的显微镜阶段或活细胞培养室视频显微镜内的文化。 </li></ol> 3，明场和视频显微镜<ol><li>含有细胞片上显微镜阶段，最初位置菜肴聚焦用4X物镜。 注：较高的放大倍数也可以使用，这取决于实验。标记的位置每个电池板和/或在舞台上的菜取向将有助于拍摄图像随着时间的推移与片材在大致相同的方向。 </li><li>去除碗碟从培养箱和照片在不同时间点，根据每个实验方案，并放置放回培养箱如果需要在实验时间帧仅静止图像。 </li><li>在执行视频显微镜，要注意以下几点： <ol><li>定位的视场，使得迁移细胞片仍然在视为视频的持续时间的字段中的尺度/新兴细胞片。 注意：这可能需要在观察细胞如何从规模下，迁移了一些经验。对于短视频，这是不到更长的录像的关注。 </li><li>对于较短的时间框架，在室温下孵育。对于较长的时间段（18+小时），使用加热阶段或孵化室要保持温度，酸碱度平衡，并尽量减少文化米蒸发edium。 </li></ol></li></ol> 4.固定的免疫荧光显微镜<ol><li>准备和预暖的所有解决方案，以28°C。为了查看角膜荧光下，上面用玻璃底的35mm组织培养皿中所描述的增长外植体文化。另外，使用玻璃玻片。 </li><li>在1.1倍PBS添加0.8毫升4％多聚甲醛的每一道菜。不先取出培养基。 在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>在1.1倍PBS添加0.8毫升4％多聚甲醛的每一道菜。在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>除非使用外部配体或外部的表位，通透细胞在1X PBS中加入0.5ml的0.2％的Triton X-100。在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>加毫升1％的0.5 BSA的1×PBS中并孵育1小时（RT）或O / N（在4℃）。 注：根据实验条件，PHAlloidin可以在这个步骤中加入。 </li><li>孵育后，吸出物，除去溶液，并继续进行抗体染色。在4℃4周 - 2毫升1％的BSA在1×PBS中进行3 - 可替代地，通过将​​至少1存储培养皿。 </li></ol> 5.免疫荧光协议<ol><li>根据制造商的建议制备初级和次级抗体溶液。 注意：如果制造商的建议都无法使用，一个良好的开端稀释的初级抗体（以在1×PBS中的1％BSA）为1：500的多克隆和1：1000用于单克隆抗体;一个良好的开端稀释的二抗是1：1000。调节从制造商处获得更高的原始股票的这些稀释液，如果信号较弱，低级如果信号强和背景是一个问题。 </li><li>吸在1×PBS溶液中的1％BSA中。加入0.5毫升初级抗体溶液孵育1小时（RT）或O / N（在4℃）。 </li&gt; <li>除去第一抗体溶液，放入一个干净的和标记的1.5 ml离心管，并冷冻在-20℃。 注意：这允许第一抗体的再利用，如果需要的话。 </li><li>与1X PBS 5次，删除和每次冲洗后丢弃的PBS - 洗碟3。 </li><li>加入0.5毫升二抗溶液。如果需要的话，在该步骤中添加荧光标记的鬼笔环肽（以下任何制造商建议的浓度）。孵育1小时（RT）或O / N（在4℃）。 注：使用荧光标记的鬼笔允许肌动蛋白丝的可视化;我们已经发现，488标记的鬼笔环肽时，最好在我们的斑马鱼角膜，但其它的荧光团都可以使用。 </li><li>取下并丢弃二抗溶液。 </li><li>与1X PBS 5次，删除和每次冲洗后丢弃的PBS - 洗碟3。 注：该盘可以存储，涵盖了从光，在4℃，如果无法争夺瓦特立刻;请确保的1×PBS被加入到培养皿存储，以确保细胞不干燥而温暖至室温之前（20 - 30分钟），然后取出PBS中继续下一步骤之前，为了。 </li><li>当准备观察，加2 - 3滴的安装介质，有或没有DAPI，根据实验需要，放入盘中，并让坐在在室温10分钟。 注意：我们使用市售的，基于甘油的安装介质用DAPI，但也可以使用其它的安装介质。允许10分钟用于安装介质坐在盘便于容易除去玻璃盖玻片从培养皿的底部。 </li><li>孵化后，轻轻挤压在菜面松动并取出盖玻片。加安装媒体的附加压降到载玻片并放置盖玻片，将细胞下来，到滑动。 注意：考虑密封盖玻片使用明确指甲油滑动如果滑动需要保持时间超过1 - 2天。 </li><li>观察在荧光显微镜下的幻灯片。 注：我们的大多数的图像被下使用油浸一个40X或63X物镜拍摄，但具体的物镜使用将取决于实验方案。 </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+and+collective+cell+migration+in+24+primary+zebrafish+explant+cultures:+MMP13+plays+an+inhibitory+role+and+MMP14+may+respond+to+stretch+during+reepithelialisation.">Matrix metalloproteinases and collective cell migration in 24 primary zebrafish explant cultures: MMP13 plays an inhibitory role and MMP14 may respond to stretch during reepithelialisation.</a> Cell Biology International Reports. 20, 24-36 (2013).</li><li>Haage, A., Nam, D. H., Ge, X., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-14+is+a+mechanically+regulated+activator+of+secreted+MMPs+and+invasion.">Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.</a> Biochemical and Biophysical Research Communications. , (2014).</li><li>Sire, J. -. Y., Girondot, M., Babiar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marking+zebrafish,+Danio+rerio+(cyprinidae),+using+scale+regeneration.">Marking zebrafish, Danio rerio (cyprinidae), using scale regeneration.</a> The Journal of Experimental Zoology. 286, 297-304 (2000).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teleost+skin,+an+ancient+mucosal+surface+that+elicits+gut-like+immune+responses.">Teleost skin, an ancient mucosal surface that elicits gut-like immune responses.</a> Proceedings of the National Academy of Sciences. 110, 13097-13102 (2013).</li><li>Keren, K., Yam, P. T., Kinkhabwala, A., Mogilner, A., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+fluid+flow+in+rapidly+moving+cells.">Intracellular fluid flow in rapidly moving cells.</a> Nature Cell Biology. 11, 1219-1224 (2009).</li><li>Bereiter-Hahn, J., Strohmeier, R., Kunzenbacher, I., Beck, K., Voth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotion+of+Xenopus+epidermis+cells+in+primary+culture.">Locomotion of Xenopus epidermis cells in primary culture.</a> Journal of Cell Science. 52, 289-311 (1981).</li><li>Nishikawa, A., Shimizu-Nishikawa, K., Miller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+in+vitro+culture+of+larval+and+adult+epidermal+cells+of+the+frog+Xenopus+laevis.">Isolation, characterization, and in vitro culture of larval and adult epidermal cells of the frog Xenopus laevis.</a> In Vitro Cellula., &amp; Developmental Biology. 26, 1128-1134 (1990).</li><li>Schober, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+kinase+modulates+tension+signaling+to+control+actin+and+focal+adhesion+dynamics.">Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics.</a> The Journal of Cell Biology. 176, 667-680 (2007).</li></ol>

作者什么都没有透露。

对于角膜移植培养的成功的最关键的步骤是使刻度附着到培养皿约2 - 除了培养基的前3分钟。大约鳞的75％将附着和生长片材（将需要建立每个实验培养物的数量，以进行相应的调整）。角膜片不形成围绕每个尺度从鱼取出，并放置在培养有几个原因。首先，外植体的DAPI染色揭示了一些鳞有少数细胞附着，因此，预计这些尺度不会有角膜的表。其次，外植体要坚持组织培养皿的能力是板材迁移的?...

<html>细胞运动性的​​研究已经传统上集中于单独迁移细胞。角膜从鱼类和两栖类植培养已证明是一个功能强大的模型系统使用实验和数学建模方法1-6来研究细胞运动的机制。上单独迁移细胞的实验工作是由细胞的快速，平稳的滑行动作的帮助下，同时保持一个统一的形状，速度和方向。然而， 在体内 ，细胞频繁移动的统称，同时保持细胞-细胞连接，尤其是在胚胎发育，伤口愈合，和转移。因此，集体细胞迁移是研究细胞迁移的领域内不断增长和日益突出的区域。这些细胞的迁移集体最近被描述7，它有很多独特的功能，这使它成为一个强大的系统来研究集体细胞迁移的伤口愈合模型。 ove_content“&gt;当秤从麻醉的成年斑马鱼弹拨，一些上皮组织保持附着到刻度的底面。当细胞培养基中加入，这些上皮细胞，或角膜，快速地迁移至自规模集体单元的外植体培养物可被看作是一种上皮伤口愈合模型中，其中细胞远离外植体，因为它们会在整个伤口床，以重新建立完整的上皮的临时基质迁移。最近的数据表明，这种体外培养模仿的许多特征在体内在成年斑马鱼伤口愈合。伤口闭合率8转换为类似于在体外系统7观察到的迁移速度，另外， 在体内伤口愈合模型中，与华法林治疗表明，止血对率没有影响愈合，而用氢化可的松治疗，以降低免疫反应不耽误表皮细胞再生8 </SUP&gt;。作为当秤从动物取出的斑马鱼不流血，止血是不是一个主要参与者。虽然我们已经发现免疫细胞外植体，我们还没有研究它们可能对集体细胞迁移的影响。 这里介绍的协议描述了如何建立斑马鱼角膜外植体培养，以确保在许多生物测定使用的一致性和可重复性。这些外植体培养在集体细胞迁移的体外模型中特别引人注目的几个原因。首先，斑马鱼角膜是建立植文化小时内使用原代细胞。因此，这些细胞未经历与主细胞9-13的通道相关联的形态和基因表达的变化。其次，该系统是一款型号为表皮细胞再生的响应研究伤人14，作为回应伤人，上皮间质潮流的一部分启动ansition（EMT）的过程就证明了基因表达的变化和细胞骨架重排14,15。因此，发生在未处理的细胞中的基因表达和运动的背景变化已被鉴定并提供了一​​个上下文来解释与治疗的变化。另外，鱼角膜和人类角质形成细胞在功能上是等效的;两者均为各自物种的初级上皮细胞和播放在上皮伤口愈合的关键作用。第三，成年斑马鱼正获得承认作为模型系统，适用于各种包括黑素瘤和其它癌症19-21，皮肤伤口愈合8,14和组织再生22-24人类疾病16-18中。 这个模型系统的技术优势也同样引人注目。可从非营利性的，集中的设施越来越多的突变和转基因株系。具体而言，斑马鱼突变项目（ZMP）在威康信托基金会桑格研究所的目标是创建在每个蛋白质编码基因的斑马鱼基因敲除等位基因。目前，他们已经突变11892个基因，基因组的大约45％，具有24088的等位基因特征。另外，ZMP接受建议，并会产生剔除装置收费。最近的基因敲除的方法在幼虫和成年斑马鱼25-28提供了灵活性，研究发育的重要基因，这些基因转基因方法是有问题的或不切实际的功能。 由于它们的非常迅速的〜145微米/小时运动速率建立培养物29后不久，测定可以迅速地完成（一般在24小时以下）。由于实验可以迅速完成，文化发展以及在室温，用几个涉及视频显微镜哺乳动物细胞迁移试验是避免相关的技术障碍。此外，在紧缩研究预算的年龄，斑马鱼肥胖是容易且廉价地维护。 外植体的结构和行为是复杂的。作为当比例尺被去除鱼不流血，这是不可能的比浅表皮层得多的规模被除去。角膜细胞似乎是主要的细胞类型的外植体时秤最初从鱼除去细胞的绝大部分出现染色用抗E-钙粘蛋白抗体14。此外，没有任何证据表明在初始培养的成纤维细胞作为判断不存在波形蛋白染色通过免疫荧光14。但是，随着反复除垢，似乎有在植众多嗜中性粒细胞（见视频1）。当暴露于LPS我们已经确定了这些细胞作为基于它们的大小，迅速地运动，并且其迁移出电池板的形态观察结果的中性粒细胞（数据未示出）。此外，这些细胞染色鲜艳的智慧h的抗体，以嗜中性粒细胞胞质因子，以及与各种次级IgG抗体，这表明它们具有在其表面上丰富FcR的（数据未示出）。多个细胞层是外植体中存在。共焦显微镜揭示邻近前缘的单个层的存在，以在靠近刻度角膜的两个以上的层，嗜中性粒细胞可见上方，下方，和之间的细胞角膜片。 在早期时间点，将细胞在多层植两极分化的边缘，引发角膜的集体的迁移，从外植体以约145微米/小时的初始速率。涵盖的外植体的面积急剧增加，从而导致细胞的片内扩散，张力，并前进前缘的下降速率。领导者和从动单元之间快速互变是在前缘的自发形成的孔的片材7内的形成和封闭过程中观察到的。由于与外植体开始的EMT过程继续，片材片段和角膜细胞失去其专业化，快速运动。于7天培养的基因表达，并与EMT一致的形态变化，伤口愈合，和炎症反应发生植正在考虑可行大约10天14。

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:320px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">35 mm Glass Bottom Culture Dishes (Poly-D Lysine Coated)&#160;</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P35GC-1.5-14-C</td>
			<td style="width:320px;">1.5 mm&#160; thickness is best for coverslip removal.&#160; 14 mm diameter provides more culture area.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Swiss Jewelers Style Forceps, Integra, Miltex 17-303</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21909-460</td>
			<td style="width:320px;">We find that narrow, fine forceps work the best for scale removal.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing

由于其独特的游动性，鱼角膜分离从外植体培养物早已被用于研究单个细胞迁移的机制。然而，当外植体建立，这些细胞还集体移动，保持了许多这使得个体角膜一个有吸引力的模型来研究移民的特点：蠕动较快速度，广泛的肌动蛋白丰富的片层具有垂直肌动线，相对恒定的速度和迁移的方向。在早期的外植体，细胞与那些迁移统称单独迁移的快速互允许的细胞 - 细胞粘连在确定迁移模式中的作用的研究，并强调迁移的两种模式之间的分子联系。在后来的外植体的细胞失去其迅速地和集体迁移能力，上皮至间质转变发生和伤口愈合和炎症相关的基因的差异表达。因此，角膜外植体可以作为一种体外模型为皮肤创伤愈合过程中发生，并且可以代表一个独特的系统来研究集体细胞迁移的机制中所定义的基因表达的变化方案的情况下的表皮细胞再生。多种突变体和转基因斑马鱼线是可用的，其允许外植体可以从鱼具有不同遗传背景的确立。这允许不同的蛋白质，这些过程中的作用，以唯一地寻址。这里所概述的协议描述一种简单而有效的方法，用于在各种相关的集体细胞迁移测定的建立这些外植体培养中使用。

如示于图1中 ，角膜的片可以观察到从规模下建立的外植体培养的小时内迁移出去。有时，单独移植角膜已经从集中迁移片剖开可以观察到。另外，作为外植体是从已被预先弹拨鱼成立，有丰富的嗜中性粒细胞通过片材迁移。在较长的文化时期，角膜板碎片为细胞进行EMT 14。 片前进的初始速率是极其迅速，但这个速率很快变慢作为细胞扩散（面积和核间?...

Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

斑马鱼角膜基质细胞的表外植迁移，并提供了一个体外模型集体细胞迁移的上皮伤口愈合的情况下机制的研究。这些协议的细节，以建立初级外植体培养在集体细胞迁移测定法使用的有效方式。

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

zebrafish-keratocyte-explants-to-study-collective-cell-migration

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  Midwestern University. 斑马鱼角膜基质细胞的表外植迁移，并提供了一个体外模型集体细胞迁移的上皮伤口愈合的情况下机制的研究。这些协议的细节，以建立初级外植体培养在集体细胞迁移测定法使用的有效方式。 

视频: Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

科研

发育生物学

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Drosophila Model to Study Wound-induced Polyploidization

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which is a result of endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation, including resistance to oncogenic growth and genotoxic stress. The challenge has been to identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster&#39;s extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP&#39;s function.

A Drosophila Model to Study Wound-induced Polyploidization

Wound-induced polyploidization is a conserved tissue repair strategy where cells grow in size instead of dividing to compensate for cell loss. Here is a detailed protocol on how to use the fruit fly as a model to measure ploidy and its genetic regulation in epithelial wound repair.

研究伤口引起的多倍体化的果蝇模型

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it ...

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.