Name: in vitro reconstitution of spatial cell contact patterns with isolated caenorhabditis elegans embryo blastomeres and adhesive polystyrene beads
Uploaded: 2019-11-26T13:30:00
Description: Watch this Scientific Journal Video about In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads at JoVE.com

我们感谢詹姆斯·普里斯和布鲁斯·鲍曼的建议，并提供C.elegans菌株，唐·莫曼，小田美祖本，和生命科学研究所成像核心设施，用于共享设备和试剂，奥伊·希罗亚苏，丽莎·费尔南多，明吉金维护C.elegans和批判性阅读我们的手稿。我们的工作得到了加拿大自然科学和工程研究理事会（NSERC）（RGPIN-2019-04442）的支持。

1. 胶粘剂聚苯乙烯珠子的制备
注：此协议不需要无菌技术。
<ol><li>在1.5 mL微离心管中称量10mg的Carboxylate改性聚苯乙烯珠子。</li>
	<li>要清洗珠子，在管中加入1 mL的2-（N-变形）甲酸（MES）缓冲液。由于MES缓冲液不含磷酸盐和醋酸盐，可降低卡博迪米德的活性，适合用于蛋白质耦合反应。涡旋管混合珠子。</li>
	<li>通过台式离心机在 2，000 x g下旋转管子 60 s。</li>
	<li>通?...

<ol>
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重建简化的细胞接触模式将使研究人员测试特定细胞接触模式在形态形成不同方面的作用。我们用这种技术来表明细胞分裂轴是由粘接珠体接触控制的。由于分轴规范对多细胞发育至关重要，有助于形成形态生成14、干细胞分裂15、16和组织平衡15、16，这种方法应该能够阐?...

在多细胞发育过程中，单个细胞的细胞行为（例如，分轴）由各种化学和物理线索指定。理解单个细胞如何解释这些信息，以及它们如何将多细胞组装作为一个紧急属性进行调节，是形态发生研究的最终目标之一。模型有机体C.elegans对细胞级形态形成调节的理解有显著贡献，如细胞极性1、细胞分裂模式1、细胞命运决定2、组织尺度调节，如神经元接线3和器官发生4、5。虽然有各种各样的遗传工具可用，组织工程方法是有限的。
在C.elegans研究中最成功的组织工程方法是经典的胚乳分离6;由于C.elegans胚胎被蛋壳和渗透屏障7包围，其去除是这种方法的主要程序之一。虽然这种爆破器隔离方法能够以简化的方式重组细胞-细胞接触，但它不允许消除不需要的线索;细胞接触仍然构成机械（例如，附着力）和化学线索，从而限制了我们完全分析线索和细胞行为之间的因果关系的能力。
本文介绍的方法使用Carboxylate改性聚苯乙烯珠子，该珠子可以共价地与包括蛋白质在内的任何胺反应分子结合为配体。特别是，我们使用一种抗胺反应形式的罗达明红-X作为配体，使珠子既可目视跟踪，又粘附在细胞上。配体分子的珠表面和原胺组的甲苯基组由水溶性甲酰胺1-乙基-3-（二甲基氨基丙基）卡博迪米（EDAC）8、9结合。获得粘合珠子允许机械提示对细胞动力学10的影响。我们已经使用这种技术来识别细胞分裂方向10所需的机械线索。

<table>
	<tbody>
		<tr>
			<td>1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>AAA1080703</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Aspirator Tube Assembly</td>
			<td>Drummond</td>
			<td>21-180-13</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans strain: N2, wild-type</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2</td>
			<td>Strain used in this study</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans strain: KSG5, genotype: zuIs45; itIs37</td>
			<td>in house</td>
			<td>KSG5</td>
			<td>Strain used in this study</td>
		</tr>
		<tr>
			<td>Calibrated Mircopipets, 10 &#181;L</td>
			<td>Drummond</td>
			<td>21-180-13</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Carboxylate-modified polystyrene beads (30 &#181;m diameter)</td>
			<td>KISKER Biotech</td>
			<td>PPS-30.0COOHP</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>CD Lipid Concentrate</td>
			<td>Life Technologies</td>
			<td>11905031</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>Clorox</td>
			<td>Clorox</td>
			<td>N. A.</td>
			<td>For the blastomere isolation. Open a new bottle when the hypochlorite treatment does not work well.</td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>In house</td>
			<td>N.A.</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Dissecting microscope: Zeiss Stemi 508 with M stand. Source of light is built-in LED. Magnification of eye piece is 10X.</td>
			<td>Carl Zeiss</td>
			<td>Stemi 508</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified One Shot, Canada origin</td>
			<td>Gibco</td>
			<td>A3160701</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>General Use and Precision Glide Hypodermic Needles, 25 gauge</td>
			<td>BD</td>
			<td>14-826AA</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Inulin</td>
			<td>Alfa Aesar</td>
			<td>AAA1842509</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>MEM Vitamin Solution (100x)</td>
			<td>Gibco</td>
			<td>11120052</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>MES (Fine White Crystals)</td>
			<td>Fisher BioReagents</td>
			<td>BP300-100</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Multitest Slide 10 Well</td>
			<td>MP Biomedicals</td>
			<td>ICN6041805</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline, 10 x Powder</td>
			<td>Fisher BioReagents</td>
			<td>BP665-1</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140148</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>Fisher BioReagents</td>
			<td>BP431-100</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Rhodamine Red-X, Succinimidyl Ester, 5-isomer</td>
			<td>Molecular Probes</td>
			<td>R6160</td>
			<td>For the bead preparation</td>
		</tr>
		<tr>
			<td>Schneider's Drosophila Sterile Medium</td>
			<td>Gibco</td>
			<td>21720024</td>
			<td>For the blastomere isolation. Work in the tissue culture hood.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Bioshop</td>
			<td>SOD001</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution, 10 N</td>
			<td>Fisher Chemical</td>
			<td>SS255-1</td>
			<td>For the blastomere isolation</td>
		</tr>
		<tr>
			<td>Spinning disk confocal microscope: Yokogawa CSU-X1, Zeiss Axiovert inverted scope, Quant EM 512 camera, 63X NA 1.4 Plan apochromat objective lens. System was controlled by Slidebook 6.0.</td>
			<td>Intelligent Imaging Innovation</td>
			<td>N.A.</td>
			<td>For live-imaging</td>
		</tr>
		<tr>
			<td>Syringe Filters, PTFE, Non-Sterile</td>
			<td>Basix</td>
			<td>13100115</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 3.175 mm</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>89403-862</td>
			<td>For the blastomere isolation.</td>
		</tr>
		<tr>
			<td>Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 6.35 mm</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>89403-854</td>
			<td>For the blastomere isolation.</td>
		</tr>
	</tbody>
</table>

in vitro reconstitution of spatial cell contact patterns with isolated caenorhabditis elegans embryo blastomeres and adhesive polystyrene beads

在多细胞系统中，单个细胞被来自邻近细胞和环境的各种物理和化学线索所包围。这种组织的复杂性混淆了外在线索和细胞动力学之间的因果关系的识别。合成重组的多细胞系统克服了这个问题，使研究人员能够测试特定的线索，同时消除其他线索。在这里，我们提出了一种方法，以重组细胞接触模式与孤立的Caenorhabditis elegans爆炸和粘胶聚苯乙烯珠。这些程序包括蛋壳去除、通过破坏细胞粘附性进行胚分裂、制备粘胶聚苯乙烯珠子以及重组细胞-细胞或细胞珠接触。最后，本文介绍了该方法在胚胎发育中研究细胞分裂轴的定位，有助于调节发育胚胎中的空间细胞模式和细胞命运规范。这种健壮、可重复和多功能的体外方法有助于研究空间细胞接触模式与细胞反应之间的直接关系。

对于珠子制备，我们确定了表达GFP-肌苷II和mCherry-组蛋白的转基因菌株的红-X琥珀酯的最佳量（图1A-D）。我们使用 mCherry 标记组蛋白作为细胞周期进展的标记。由于罗达明红X-X和mCherry将由561nm激光照明，因此罗达明红X-X信号的最佳强度与组蛋白相当，可以同时成像细胞和珠子。例如，使用 0.005 μg/mL Rhodamine 红-X 琥珀酯处理的珠子的荧光信号太弱，无法可视化珠（...

Watch this Scientific Journal Video about In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads at JoVE.com

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

多细胞系统的组织复杂性混淆了细胞外线索与单个细胞行为之间的因果关系的识别。在这里，我们提出了一种方法，研究接触依赖线索和分裂轴之间的直接联系使用C.elegans胚胎胚芽和粘合聚苯乙烯珠子。

在Vitro重建空间细胞接触模式与孤立的Caenorhabditis elegans胚胎冲击器和粘性聚苯乙烯珠

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

多细胞组织复杂，此协议以简化的方式重组细胞行为。有一件事是这种技术来研究细胞导体质子和细胞反应之间的直接关系。该协议使用化学功能聚苯乙烯珠子来重组细胞导体质子。珠子可以与任何感兴趣的质子切割，以测试它们对细胞反应的影响。我们在小鼠胚胎上的C-优雅中使用过这种方法。因此，该协议可以直立于对广泛生物的研究。由于胚胎在高全氯处理后很脆弱，因此在成功分离异丙酮之前，需要大量实践。视觉演示将加深对在风波分离方法期间如何处理毛细管的理解。首先，在1.5毫升微型离心管中，重达10毫升的碳氧酸酯改性聚苯乙烯珠。将一毫升 MES 缓冲液加入管中进行清洗。旋转管混合珠子。通过台式离心机在 2000XG 下旋转管 60 秒。通过小心地移出缓冲液，将上看液去掉。用一毫升的 MES 缓冲液再次清洗珠子。洗涤后，将含有10毫升EDAC的1毫升 MES 缓冲液加入管中，激活表面的箱状组。旋转管混合珠子。在室温下旋转并孵育管子15分钟。在 2000XG 下旋转珠子 60 秒。通过小心地将缓冲液点出，用一毫升 PBS 清洗，丢弃上流水线。漩涡管混合珠子，重复PBS洗涤一次。从 0.65 毫升罗达明红-X 库存溶液中准备一毫升的一升、10、100 和千倍稀释系列的罗达明红-X。皮佩特20微升珠到每个串行稀释管。在室温下旋转并孵育管子五分钟。用一毫升 PBS 清洗珠子两次。洗涤后，将一毫升PBS加入管子，并在4摄氏度下储存长达六周。在用于实时成像的显微镜下检查珠子的荧光强度。用右手和左手将微型毛细管的每一端握住，容量为 10 微升。将微毛细管拉向两端，以施加张力，并将毛细管的中心放在燃烧器上，使两个手动拉毛细管。在解剖显微镜下，用钳子修剪手拉毛细管的尖端。使两个尖端开口大小大约两倍，并分别是用于胚胎移植和蛋壳去除的C-优雅胚胎的短访问长度的一倍。将拉扯的毛细管连接到口蹄疫装置中。Pippette 45 微升蛋盐溶液放到多井滑梯的井上。将5至10名成人C-优雅放在井上。为了获得早期的C-优雅胚胎，通过将两根针定位到C-Elegance身体的左右两根针，然后互相滑动针头，将成人切成碎片。皮佩特45微升次氯酸盐溶液到井旁边的井含有蛋盐溶液和皮佩特45微升谢尔顿的生长介质到随后的三口井。将一个细胞阶段和早期两个细胞阶段胚胎转移到次氯酸盐溶液中，用较大的开口，等待40至55秒。用谢尔顿的生长介质将胚胎移植到接下来的三口井中，以清洗胚胎。前两口井各有一到两秒。使用手绘毛细管与较小的开口仔细重复管道胚胎的蛋壳去除。之后，用手绘毛细管连续分离双细胞阶段胚胎胚胎。去除蛋壳后，尽量减少上下对胚胎进行管道的力，以防止破裂。通过将盖玻片放在盖玻片支架上，并胶带固定盖玻片的边缘来稳定其，来准备成像室。翻转盖玻片支架，用疏水笔在盖玻片上画一个圆圈。在疏水标记绘制的圆内添加 Shelton 的生长介质，并将隔离的爆炸体转移到圆内的成像室。现在，使用具有较大开口的手绘毛细管将少量化学功能化珠子分配到盖玻片上。通过吹入手绘毛细管，直到珠子附着在孤立的气管上，控制珠子的位置。安装盖玻片，以避免介质蒸发，并使用倒置显微镜进行实时成像。在这项研究中，用0.005微克处理的珠子的荧光信号，用于表达GP miosin-2和M-cherihistone的转基因菌株。另一方面，从珠子处理五微克每毫升罗达明红-X苏西尼米基酯的荧光信号太强。罗达明红-X苏西尼米基酯的浓度为0.5微克每毫升是最佳的这种特殊的转基因菌株。在实时成像过程中，识别到两个中心垂直对齐的平面。此平面的正负 90 度角的平面是主轴平面。细胞因子后相对于细胞/珠接触界面的主轴方向角度显示，两个子细胞在通过两个接触眼的线中连接到珠子，用作接触方向。人们需要了解胚胎和蛋壳对嘴嘴皮盘施加的某些外力的反应。从理论上讲，任何细胞反应都可以研究，例如，硫酸盐规格可以通过培养细胞和接触胶粘珠长期研究。此技术已用于了解接触依赖细胞分裂方向机制。PTF过滤器用于防止通过口蹄疫吸入次氯酸盐溶液的烟雾。

Research

JoVE Journal

Developmental Biology

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...