Name: xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization
Uploaded: 2021-06-06T13:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

我们感谢J. Kamenz、Y. Chen和W. Y. C. Huang对手稿的评论。这项工作得到了美国国立卫生研究院（R01 GM110564，P50 GM107615和R35 GM131792）的资助，授予James E. Ferrell，Jr.。

此处描述的所有方法均已获得斯坦福大学机构动物护理和使用委员会（IACUC）的批准。
1. 载玻片和盖玻片的准备
<ol><li>用滚筒涂抹器将一层氟化乙烯丙烯（FEP）胶带涂在载玻片上。用干净的剃须刀片剪掉边缘上多余的胶带。以相同的方式制备FEP胶带包被的盖玻片（图1A）。</li>
	<li>将双面粘性成像垫片涂在载玻片的FEP胶带涂层面上。保持顶部的保护衬垫...

<ol>
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非洲爪蟾卵提取物已成为一种强大的模型系统，用于各种亚细胞结构的成像研究10，14，15，16，17，18，21，31，32，33，34，35<su...

细胞质构成细胞的主要体积，具有独特的组织。真核细胞质的成分可以自组装成广泛的空间结构，例如微管紫苑和高尔基体，而微管紫苑又根据细胞的身份和生理状态动态排列和翻转。因此，了解细胞质的空间组织及其与细胞功能的联系对于理解细胞如何工作非常重要。非洲爪蟾卵提取物传统上用于批量生化测定1，2，3，4，5，6，7，8，但最近的工作将它们确立为强大的实时成像系统，用于细胞质结构及其细胞功能的机制研究9，10，11，12，13，14，15，16，17，18.这些未稀释的提取物保留了细胞质的许多结构和功能，同时允许直接操作细胞质内容物，这是传统基于细胞的模型无法实现的19，20。这使得它们成为表征细胞质现象和剖析其机制基础的理想选择。
现有的提取物成像方法需要化学表面改性或制造微流体装置。一种基于盖玻片的方法需要对玻璃盖玻片进行聚乙二醇（PEG）钝化21。基于微乳液的方法需要在玻璃表面上气相沉积三氯（1H，1H，2H，2H-全氟辛基）硅烷22，23。基于微流体的系统可以精确控制提取物液滴的体积、几何形状和组成，但需要专门的微细加工设施11，12，24。
在这里，我们介绍了一种对卵子提取物进行成像的替代方法，该方法易于实施并利用现成的低成本材料。这包括制备带有载玻片和涂有氟化乙烯丙烯（FEP）胶带的盖玻片的成像室。该腔室可用于使用各种显微镜系统对提取物进行成像，包括立体镜以及正置和倒置显微镜。该方法不需要对表面进行化学处理，同时获得与上述现有玻璃方法相似的光学透明度。它旨在对 2D 视场中厚度均匀的提取物层进行成像，并且可以轻松扩展以对提取物的 3D 体积进行成像。它非常适合在大视场上对集体细胞质行为进行延时成像。
我们使用相间停滞的卵提取物来演示我们的成像方法。提取物制备遵循戴明和科恩布鲁斯19的方案。简而言之，在减数分裂II中期自然停滞的卵子被低速旋转压碎。该旋转将细胞质从减数分裂停滞中释放出来，并允许提取物进入间期。通常，在粉碎自旋之前添加细胞松弛素B以抑制F-肌动蛋白的形成。然而， 如果需要 F-肌动蛋白， 它可以省略.在粉碎旋转之前还添加环己酰亚胺，以防止中间提取物进入下一个有丝分裂。随后将提取物置于上述成像室中并置于显微镜上。最后，通过连接到显微镜的相机以定义的时间间隔记录图像，生成延时图像系列，在2D场中捕获提取物的动态行为。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17 ml centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>337986</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm square #1 cover glass</td>
			<td>Corning</td>
			<td>284522</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>MilliporeSigma</td>
			<td>10236624001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>MilliporeSigma</td>
			<td>01810</td>
			<td>Protein synthesis inhibitor</td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>MilliporeSigma</td>
			<td>C6762</td>
			<td>Actin polymerization inhibitor</td>
		</tr>
		<tr>
			<td>Female Xenopus laevis frogs</td>
			<td>Nasco</td>
			<td>LM00535MX</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 488 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL488M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 647 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL670M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorinated ethylene propylene (FEP) optically clear tape</td>
			<td>CS Hyde company</td>
			<td>23-FEP-2-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (hCG)</td>
			<td>MilliporeSigma</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging spacer</td>
			<td>Electron Microscopy Sciences</td>
			<td>70327-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>MilliporeSigma</td>
			<td>11017101001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>MilliporeSigma</td>
			<td>330760</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CMXRos</td>
			<td>Thermo Fisher Scientific</td>
			<td>M7512</td>
			<td>For visualizing mitochondria</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>BioVendor</td>
			<td>RP1782725000</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller applicator</td>
			<td>Amazon</td>
			<td>B07HMBJSP8</td>
			<td>For applying the FEP tape to the glass slides and coverslips</td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>For removing excessive FEP tape</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
	</tbody>
</table>

xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization

非洲 爪蟾 卵提取物传统上用于批量生化测定，已成为一种强大的基于成像的工具，用于研究细胞质现象，例如细胞分裂、有丝分裂纺锤体形成和细胞核组装。基于对在稀疏时间点采样的固定提取物进行成像的早期方法，最近的方法使用延时显微镜对实时提取物进行成像，揭示了具有增强时间分辨率的更多动态特征。这些方法通常需要对成像容器进行复杂的表面处理。在这里，我们介绍了一种无需化学表面处理的鸡蛋提取物实时成像的替代方法。它易于实施，并利用批量生产的实验室耗材进行成像。我们描述了一种可用于宽场和共聚焦显微镜的系统。它专为二维（2D）场中的提取物成像而设计，但可以轻松扩展到3D成像。它非常适合研究细胞质内的空间模式形成。通过代表性数据，我们证明了使用该方法制备的相间提取物中微管，细胞核和线粒体的典型动态组织。这些图像数据可以提供有关细胞质动力学和空间组织的定量信息。

非洲爪蟾卵提取物可用于研究间期细胞质的自组织。图2A显示了成功实验的结果。我们用浓度为 27 个细胞核/μL 的去膜爪蟾精子核19 和 0.38 μM 纯化的 GST-GFP-NLS27、28、29、30（由谷胱甘肽-S-转移酶、绿色荧光蛋白和核定位序列组成的?...

Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

我们描述了一种从 非洲爪蟾 卵中制备和实时成像未稀释的细胞质提取物的方法。

非洲爪蟾 用于可视化动态细胞质组织的蛋提取物制备和实时成像方法

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic ...

该方法适用于使用青蛙卵提取物对细胞质动力学进行实时成像，从而能够研究细胞质中的空间模式信息。样品可以塑造成厚度均匀且可调节的层，并在 2D 视场或 3D 体积上成像。这种方法具有成本效益且易于实施。首先用滚筒涂抹器将一层FEP胶带涂在载玻片上。用干净的剃须刀片剪掉边缘上多余的胶带。接下来，以相同的方式准备 FEP 胶带涂层的盖玻片。然后将双面粘性成像垫片涂在载玻片的FEP胶带涂层面上，保持保护衬里不剥落。将鸡蛋转移到 400 毫升玻璃烧杯中，并通过倾析尽可能多地去除产卵缓冲液。然后将鸡蛋在 100 毫升新鲜制备的脱冻溶液中孵化并轻轻旋转。约三分钟后，倒出溶液，加入100毫升新鲜果冻溶液。继续孵育，直到鸡蛋紧密包装，但避免将鸡蛋留在脱冻溶液中超过五分钟。孵育后，尽可能去除脱胶溶液，并在0.25x MMR缓冲液中洗涤鸡蛋。旋转鸡蛋，然后倒掉缓冲液。重复几次，直到总共使用一升缓冲液进行洗涤。接下来，用总共400毫升的鸡蛋裂解缓冲液清洗鸡蛋几次，在洗涤之间去除异常鸡蛋。然后使用带有宽切尖端的转移移液器将鸡蛋转移到含有一毫升鸡蛋裂解缓冲液的 17 毫升圆底离心管中。在临床离心机中以400g旋转试管15秒以包装鸡蛋。然后使用巴斯德移液管尽可能多地去除鸡蛋裂解缓冲液。确定包装鸡蛋的大致体积。然后直接在包装好的鸡蛋上添加每毫升 5 微克抑肽酶、亮肽酶和细胞松弛素 B 以及每毫升 50 微克环己酰胺。通过在摆动桶转子中以 12， 000 克和 4 摄氏度离心管 15 分钟来压碎鸡蛋。接下来，将18号针头连接到注射器上。针尖斜面朝上，从细胞质层底部的一侧刺穿管子，并通过缓慢抽取提取物来回收提取物。将回收的细胞质提取物转移到新的微量离心管中，并将其保存在冰上。准备好成像后，用所需的试剂和荧光成像探针补充提取物。从准备好的载玻片上的成像垫片上取下顶部保护衬里，并在孔的中心沉积约7微升提取物。立即使用FEP胶带涂层盖玻片，FEP面朝向提取物以密封孔并快速进行成像。将载玻片放在带有电动载物台和数码相机的倒置或正置显微镜上。在明场和荧光通道中以时间间隔在所需的空间位置对提取物进行成像。这里显示了使用非洲爪蟾卵提取物研究间期细胞质自组织的延时实验。在室温下孵育约20分钟后，在明场和线粒体通道中都可以看到光散射细胞质成分耗尽的区域。在室温下约60分钟，由细胞样隔室组成的空间模式应该很好地建立，微管形成空心花环状结构，线粒体明确划分到每个隔室中。此处显示了在玻璃上使用和不使用FEP胶带的成像室中提取物性能的比较。在用FEP胶带玻璃制成的腔室中，提取物自组织成正常的细胞样图案。在玻璃表面未被FEP胶带覆盖的腔室中，提取物显示出异常的明场和微管图案，随着时间的推移变得越来越混乱。在GST-mCherry-NLS蛋白的核输入方面没有观察到显着差异。要记住的重要事项是用FEP胶带钝化玻璃表面，并在提取物制备过程中去除所有坏蛋。有了这个系统，我们可以轻松控制提取物样品的厚度，为使用光片显微镜对细胞质图案进行3D成像铺平了道路。

Research

JoVE Journal

Biology

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Plastic Embedding and Sectioning of Xenopus laevis Embryos

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparing Individual Drosophila Egg Chambers for Live Imaging

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Reconstitution Of ÃƒÅ½Ã‚Â²-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

非洲爪蟾 用于可视化动态细胞质组织的蛋提取物制备和实时成像方法

非洲爪蟾用于可视化动态细胞质组织的蛋提取物制备和实时成像方法