Name: a 3-dimensional (3d)-printed template for high throughput zebrafish embryo arraying
Uploaded: 2018-06-01T15:00:00
Description: Watch this Scientific Journal Video about A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying at JoVE.com

这项工作得到了 "1000plan 青年" 计划的支持, 同济大学的创业基金, 以及21607115和21777116的自然科学基金 (Lin)。

1. 斑马鱼胚胎排列模板的设计与制作
<ol><li>
设计排列模板的 12, 8, 96 井布局, 适合一个标准的96井板。使用图 1A中列出的用于上胚卡压室的尺寸 (请参阅补充文件)。
	<ol>
<li>使用在图 1B和1D中显示的用于陷井的尺寸。</li>
		<li>使用底部真空室的图 1C中的尺寸。</li>
		<li>将图 1B中的尺?...

<ol>
	<li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Leslie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+larvae+could+help+to+personalize+cancer+treatments.">Zebrafish larvae could help to personalize cancer treatments.</a> Science. 357 (6353), 745-745 (2017).</li><li>Lin, S., 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=Understanding+the+Transformation,+Speciation,+and+Hazard+Potential+of+Copper+Particles+in+a+Model+Septic+Tank+System+Using+Zebrafish+to+Monitor+the+Effluent.">Understanding the Transformation, Speciation, and Hazard Potential of Copper Particles in a Model Septic Tank System Using Zebrafish to Monitor the Effluent.</a> ACS Nano. 9 (2), 2038-2048 (2015).</li><li>Lin, S., 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=Aspect+ratio+plays+a+role+in+the+hazard+potential+of+ceo2+nanoparticles+in+mouse+lung+and+zebrafish+gastrointestinal+tract.">Aspect ratio plays a role in the hazard potential of ceo2 nanoparticles in mouse lung and zebrafish gastrointestinal tract.</a> ACS Nano. 8 (5), 4450-4464 (2014).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nature Communications. 4, (2013).</li><li>Lin, S., 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=High+content+screening+in+zebrafish+speeds+up+hazard+ranking+of+transition+metal+oxide+nanoparticles.">High content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles.</a> ACS Nano. 5 (9), 7284-7295 (2011).</li><li>Kuipers, J., Kalicharan, R. D., Wolters, A. H. G., van Ham, T. J., Giepmans, B. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+Scanning+Transmission+electron+microscopy+(nanotomy)+of+healthy+and+injured+zebrafish+brain.">Large-scale Scanning Transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.</a> Journal of Visualized Experiments. (111), (2016).</li><li>Liu, R., 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=Automated+Phenotype+Recognition+for+Zebrafish+Embryo+Based+In+vivo+High+Throughput+Toxicity+Screening+of+Engineered+Nano-Materials.">Automated Phenotype Recognition for Zebrafish Embryo Based In vivo High Throughput Toxicity Screening of Engineered Nano-Materials.</a> PLoS One. 7 (4), (2012).</li><li>Tu, X., 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=Automatic+Categorization+and+Scoring+of+Solid,+Part-Solid+and+Non-Solid+Pulmonary+Nodules.">Automatic Categorization and Scoring of Solid, Part-Solid and Non-Solid Pulmonary Nodules.</a> in CT Images with Convolutional Neural Network. Scientific Reports. 7, 8533 (2017).</li><li>Mandrell, D., 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=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Lin, S., Zhao, Y., Nel, A. E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+in+vivo+model+for+nano+EHS+studies.">Zebrafish: An in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li></ol>

该协议中有两个关键步骤需要密切注意, 以成功地实施 3 d 打印模板排列斑马鱼胚胎。
排列模板设计中最重要的因素是诱捕井。为了确保每个井中只有一个胚胎被困, 人们应该密切关注压井的直径和深度, 以及通孔的直径。建议的直径在一个典型的胚胎 (包括绒毛膜) 直径的1.5 到2倍之内。诱捕井的深度应在典型胚胎 (包括绒毛膜) 直径的2倍以内, 以避免在同一井中堆积胚胎。通?...

斑马鱼作为一种流行的模型生物, 广泛应用于医学和毒物学领域1,2,3,4。与体外平台相比, 斑马鱼提供了更大的生物复杂性, 即一个或两种细胞无法提供。除了作为一个整体的生物模型, 斑马鱼的巨大繁殖力, 快速和同时胚胎发育, 和高器官半透明已经给这个模型独特的优势, 用于大规模毒性或药物/复合筛查5。一双成年斑马鱼每周生产的数以百计的胚胎超过了其他所有的动物模型, 并使其适合高通量筛选。
一个典型的筛选程序使用斑马鱼涉及大量的手工工作, 如成年斑马鱼产卵, 胚胎选择, 并排列胚胎进入适当的容器, 他们通过水浸泡暴露。对胚胎的发育进行监测, 可观察到的端点, 如死亡率、孵化率和异常, 通常是手动评估的, 并作为化学品毒性的初步鉴定或指示药物或化合物。为了加快筛选程序, 前面已经探讨了自动成像和计算机辅助图像分析等方法。例如, 具有高含量成像能力的显微镜已经适应了从96/384 井板6的不同发育阶段对斑马鱼胚胎进行自动亮场或荧光成像。微流控装置与显微镜结合使用, 用于定位斑马鱼幼虫通过当前操作的大脑神经元的成像7。与传统的手工操作相比, 这些方法可以显著提高图像获取的效率。此外, 在生成大量图像的同时, 还开发了图像分析工具以加快数据处理速度, 如刘et和 Tu et . 所示。8,9。
随着成像和图像分析的吞吐量水平的提高, 显而易见的是, 筛查速率限制的步骤在于准备用于暴露的斑马鱼胚胎, 这通常意味着将它们排列到96或384井板中。为解决这一瓶颈步骤, Mandrell 开发了视觉导引机器人. 10和美国11以前替换手动处理, 但这些工具相当复杂, 并且有一个深入的学习曲线来实现这些技术。因此, 提供一种易于使用的方法成为进一步提高斑马鱼筛查吞吐量水平的一个重要因素, 是这项工作的主要目标。
在这项工作中, 我们设计和制造了一个胚胎排列模板3D 打印。这种排列模板的设计, 以诱捕斑马鱼胚胎进入水井, 符合标准的96井板。而不是选择胚胎和排列他们一个一个一个一个, 你可以执行胚胎诱捕和阵列所有96胚胎成一个多层板一次。使用这个模板和下面的协议, 你可以显著提高排列胚胎的效率到多层板, 这将在短期内提高筛选能力至少比手工操作。下面描述的协议包括排列模板、斑马鱼产卵、胚胎采集和排列的总体设计。图 1显示了排列模板的总体设计。图 2显示了使用部件3和4中描述的模板的分步协议概述。

<table border="0" cellpadding="0" cellspacing="0" width="1557">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Zebrafish Facility</td>
			<td style="width:425px;">Shanghai Haisheng Biotech Co., Ltd.</td>
			<td style="width:229px;">Z-A-S5</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mating box</td>
			<td style="width:425px;">Shanghai Haisheng Biotech Co., Ltd.</td>
			<td style="width:229px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Wash Bottle, 500 ml</td>
			<td style="width:425px;">Sangon Biotech</td>
			<td style="width:229px;">F505001-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:425px;">Vetec</td>
			<td style="width:229px;">V900058-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td style="width:425px;">Sinopharm Chemical Reagent Co.,Ltd</td>
			<td style="width:229px;">10016318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td style="width:425px;">Sinopharm Chemical Reagent Co.,Ltd</td>
			<td style="width:229px;">20011160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate&#160;</td>
			<td style="width:425px;">Vetec</td>
			<td style="width:229px;">v900182-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue Hydrate</td>
			<td style="width:425px;">TCI</td>
			<td style="width:229px;">M0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:425px;">Sinopharm Chemical Reagent Co.,Ltd</td>
			<td style="width:229px;">10011008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sea Salts</td>
			<td>Instant Ocean</td>
			<td style="width:229px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetter</td>
			<td style="width:425px;">Fisherbrand</td>
			<td style="width:229px;">13-675M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Controlled Drop Pasteur Pipet</td>
			<td style="width:425px;">Fisherbrand</td>
			<td style="width:229px;">13-678-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope</td>
			<td style="width:425px;">OLYMPUS</td>
			<td style="width:229px;">SZ61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biochemical incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>LRH-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D printer</td>
			<td>UnionTech</td>
			<td>Lite600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photosensitive resin</td>
			<td>UnionTech</td>
			<td>UTR9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum pump</td>
			<td>Shanghai Yukang Scientific Instrument Co., Ltd.</td>
			<td>SHB-IIIA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Adhesive PCR Plate Seals</td>
			<td style="width:425px;">Solarbio</td>
			<td style="width:229px;">YA0245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well plate</td>
			<td style="width:425px;">Costar</td>
			<td style="width:229px;">3599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Multi 8-channel pipette 30 - 300 &#956;l</td>
			<td style="width:425px;">Eppendorf</td>
			<td style="width:229px;">3122000.051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Compressed Gas Duster</td>
			<td style="width:425px;">Shanghai Zhantu Chemical Co., Ltd.</td>
			<td style="width:229px;">ST1005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DI Water</td>
			<td style="width:425px;">Thermo</td>
			<td style="width:229px;">GenPure Pro UV/UF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Drying oven</td>
			<td style="width:425px;">Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td style="width:229px;">BPG-9106A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">System water</td>
			<td style="width:425px;"></td>
			<td style="width:229px;"></td>
			<td style="width:429px;">Water out of the facility&#8217;s water system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">Egg water</td>
			<td style="width:425px;"></td>
			<td style="width:229px;"></td>
			<td style="width:429px;">Dilute 60mg &#8220;Instant Ocean&#8221; sea salts and 0.25 mg/L methylene blue in 1 L DI water</td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:475px;">Holtfreter&#8217;s solution</td>
			<td style="width:425px;"></td>
			<td style="width:229px;"></td>
			<td style="width:429px;">Dissolve 7.0 g Sodium chloride (NaCl), 0.4 g Sodium bicarbonate (NaHCO3), 0.1 g Potassium Chloride (KCl), 0.235 g Calcium chloride (CaCl2.2H2O) in 1.9 L DI water. Adjust pH to 7 using HCl and adjust volume to 2 L using Di water</td>
		</tr>
	</tbody>
</table>

a 3-dimensional (3d)-printed template for high throughput zebrafish embryo arraying

斑马鱼是全球公认的淡水有机体, 经常用于发展生物学、环境毒理学和人类疾病相关的研究领域。由于其独特的特点, 包括大的繁殖力, 胚胎半透明, 快速和同时发展,等, 斑马鱼胚胎经常被用于对化学品进行大规模毒性评估和药物/复合筛查。一个典型的筛选程序涉及成年斑马鱼产卵, 胚胎选择, 并排列胚胎成多井板块。从那里, 胚胎受到接触和化学的毒性, 或者药物/化合物的有效性可以根据表型观察相对快速地评估。在这些过程中, 胚胎排列是限制吞吐量水平的最耗时和最耗费劳力的步骤之一。在本协议中, 我们提出了一种创新的方法, 使用一个 3 d 打印排列模板加上真空操作, 以加快这一艰巨的步骤。此处的协议描述了排列模板的总体设计, 详细的实验设置和分步过程, 随后有代表性的结果。在实施时, 这种方法应证明是有益的, 以斑马鱼胚胎作为测试对象的各种研究应用。

图 3显示了典型的 3 d 打印的排列模板。本模板采用光敏树脂为原料, 由3D 打印机制成;用一层黑色颜料, 可以更好地对比胚胎的颜色。96口井 (12 8) 的位置设计为符合标准96井板。同样, 384 (24 乘 16) 井模板也可以使用相同的方法设计和制作。向上的房间稍大于标准的96井板, 以提供一个更好的配件。凹槽也被设计用来在排列期间容纳额外的胚胎。
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Watch this Scientific Journal Video about A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying at JoVE.com

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

在这里, 我们提出了一个设计和制造斑马鱼胚胎排列模板的协议, 然后详细的程序, 使用这种模板, 高吞吐量斑马鱼胚胎排列成96井板。

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

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Evaluation of the Productivity of Social Wasp Colonies Vespinae and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

社会黄蜂群落（韦皮纳）生产力评价及日本传统黄蜂狩猎技术简介

For vespine wasps, colony productivity is typically estimated by counting the number of larval cells. This paper presents an improved method that enables ...