Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

这项工作得到了康诺特基金新调查员奖、加拿大创新基金会、NSERC发现赠款计划（授予RGPIN-2015-05114和RGPIN-2020-05881）、曼彻斯特大学和多伦多大学联合研究基金以及多伦多大学XSeed项目的支持。

1. 细胞播种
注：描述的细胞扩散协议使用小鼠胚胎成纤维细胞（MEF）执行，表达PH-Akt-GFP（PIP3/PI（3，4）P 2的荧光标记）。该细胞系通过CRISPR介质基因编辑，通过基因学整合PH-Akt-GFP（添加基因#21218）的表达结构而生成。然而，其他在基因组中短暂表达或集成的荧光标记也可以用于此测定。为了实现最佳图像分割，我们建议使用细胞质均匀分布的荧光标记，例...

<ol>
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描述的细胞扩散检测允许连续跟踪形态变化（如细胞大小和形状）和细胞边缘运动（即突起速度和缩回频率），这是大多数细胞扩散协议19，24中缺少的功能。虽然常用的端点细胞扩散检测允许确定细胞扩散速度，但这些测定无法解决细胞边缘运动的时间动力学。缺乏时间信息限制了检测和量化跛脚突起-缩回周期变化的能力。
<p class="jov...

拉梅利波德突起是生长细胞前部形成的突出细胞骨结构。在lamilipodia，在Arp2/3复合物的帮助下，行为物的聚合和福明斯创造了一个快速生长的分支行为网格，推动对等离子膜1，2。网格工程产生的推动力在物理上推动细胞向前推进1，3，4，5。Arp2/3复杂或信号通路的耗竭，对于跛脚突出至关重要，通常会损害细胞迁移6，7。虽然跛脚足缺细胞的迁移也已报告8，9，跛脚足在细胞迁移中的重要性是显而易见的，因为这种突起结构的耗竭扰动细胞通过复杂的生物微环境移动的能力6，10。
理解迁移细胞中跛脚足的调控的一个主要障碍是跛脚突出动力学的自然变异性、大小和形状11、12、13、14。此外，最近的研究表明，跛脚皮迪亚表现出复杂的突出行为，包括波动，周期性，加速突出14，15。与迁移细胞6、16的高度可变的跛脚足症相比，在细胞扩散过程中形成的跛脚足足12更为均匀。由于扩散和迁移细胞的突起活动是由相同的大分子组件驱动的，包括分支作用网络、收缩性活体蛋白束和基于内格林的细胞基质粘附17、18，因此扩散细胞被广泛用作研究跛脚体动力学调控的模型。
细胞扩散是一个动态的机械化学过程，悬浮中的细胞首先通过基于内格林的附着力17、19、20粘附粘附，然后通过延长基于作用的突起21、22、23进行扩散。在扩散阶段，从细胞体中喷出的跛脚足部突出异位和持久，很少或根本没有收回或停滞12。最常用的细胞扩散协议是端点检测，其中扩散细胞在电镀19，24后在不同时间固定。这些检测虽然快速而简单，但检测跛脚皮迪亚动态特征的变化的诊断能力有限。为了确定控制跛脚足动力学的分子机制，Sheetz小组率先对活扩散细胞进行了定量分析，并发现了细胞边缘突起11、12、22的许多基本特性。这些研究表明，活细胞传播检测是细胞生物学实验室工具箱中一种强大而有力的技术。尽管如此，细胞生物学界目前还无法为活细胞传播检测提供详细的协议和开源计算工具。为此，我们的协议概述了成像活传播细胞的程序，并提供了一个自动化的图像分析工具。为了验证这种方法，我们使用Arp2/3抑制作为实验治疗，并表明抑制Arp2/3复合体的功能不会阻止细胞扩散，但会导致细胞突起速度显著降低，以及细胞边缘突起的稳定性，从而产生锯齿状细胞边缘。这些数据表明，活细胞成像和自动图像分析的结合是分析细胞边缘动力学和识别调节跛脚皮迪亚的分子组件的有用工具。

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		<tr>
			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red</td>
			<td>Wisent Bioproducts</td>
			<td>319-005 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil</td>
			<td>Cargille</td>
			<td>16241</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution (200 mM)</td>
			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
			<td>VWR</td>
			<td>CA48366-227-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Body, Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
			<td>Nikon</td>
			<td>MRD01605</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk, Crest Light V2</td>
			<td>CrestOptics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, Style 2</td>
			<td>Electron Microscopy Sciences</td>
			<td>78326-42</td>
			<td></td>
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</table>

quantitative analysis of cell edge dynamics during cell spreading

细胞扩散是一个动态过程，其中悬浮在介质中的细胞附着在基材上，并把自己从圆形压扁成薄而分散的形状。在细胞基底附着物之后，细胞形成一块从细胞体中散发出的薄薄的跛脚状。在lamellipodia中，球状作用素（G-actin）单体聚合成密集的丝状活性蛋白（F-actin）网状物，推动对等离子膜，从而提供细胞扩散所需的机械力。值得注意的是，控制跛脚细胞聚合的分子分子对许多其他细胞过程至关重要，如细胞迁移和内分泌。 
由于扩散细胞形成横跨整个细胞外围并持续向外扩张的连续跛脚状体，细胞扩散检测已成为评估跛脚突起动力学的有效工具。虽然已经制定了细胞传播检测的若干技术实施，但目前缺乏对工作流程的详细描述，其中包括分步协议和数据分析的计算工具。在这里，我们描述了细胞扩散检测的实验过程，并提出了一个开源工具，用于定量和公正地分析细胞边缘动力学在扩散过程中。当与药理学操作和/或基因沉默技术相结合时，此协议可适应调节跛脚突出的分子参与者的大规模屏幕。

上述协议描述了扩散细胞活细胞成像的实验程序，以及用于细胞扩散动力学定量分析的计算工具。计算工具可以采用低通量或高通量格式，以识别调节细胞前沿作用聚合机械的分子玩家。
实验程序的示意图表示在图1中描述。细胞扩散检测是在不朽的小鼠胚胎成纤维细胞上进行的，稳定地表达了标有eGFP25的Akt蛋白激酶的丛斯特林同源（P...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

在此协议中，我们介绍了基于活细胞显微镜的细胞扩散检测的实验程序。我们为荧光标记细胞的无偏见分割和细胞扩散过程中的跛脚足动力学定量分析提供了开源计算工具。

细胞扩散过程中细胞边缘动力学的定量分析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

本视频中描述的协议将定义传播细胞的真人大小成像所需的实验程序，以及使用计算工具，以公正和自动化的方式量化传播动态的细胞。细胞扩散资产允许在细胞扩散过程中持续跟踪细胞边缘运动和形态变化，这是大多数现有细胞扩散检测中缺失的一个特征。同时，本协议还实现了计算机自动处理和分析的使用，提供了关于细胞循环、区域和突出缩还周期的信息。这种自动处理减少了数据分析中的偏差，并为大量细胞提供了分析细胞传播动力学的可靠方法。当与药物治疗或基因科学和技术相结合时，此协议可适应调节细胞突起的分子参与者的大规模速度。对于给定协议，使用的细胞是小鼠胚胎成纤维细胞，通过基因编码PH-Akt-GFP，允许荧光标记等离子膜。在细胞展开检测之前，培养一盘细胞达到90%的共性。一旦细胞达到适当的对流，火焰22乘22毫米覆盖滑动，并将其放入35毫米细胞培养盘。涂上在PBS中稀释的纤维素，使盖片的浓度为每毫升2.5微克。将带有盖子的盘子放入 37 度孵化器中一小时。一小时后，吸气纤维素，确保不要触摸与移液器尖端的盖滑。用PBS洗碗，轻轻在盖子周围吹两到三次。对于细胞播种，首先从细胞盘中吸气细胞培养介质。之后，用温暖的PBS洗碗。在菜中加入 650 微升的 trypsin EDTA，使菜向倾斜，以均匀地分配酶。将带试剂的菜放入孵化器中一分钟。孵化后，您首先需要将 10 毫升细胞培养介质添加到离心管中。接下来，快速在菜中加入另外10毫升的介质，以淬灭试穿素。为了稀释将播种到盖滑上的细胞，将一毫升的菜肴内容放入离心管中。从管子上，移液器约500至1000微升稀释细胞进入菜中，盖滑。确保盖滑在 10% 的汇流或每毫升 5 万个细胞，并根据需要调整稀释细胞的体积。这些细胞的目的是在图像采集过程中建立一个焦点平面。在盘中剩余的细胞中，每次处理将五分之一的细胞放入一个小盘子中。这些细胞将被分析为传播动力学。将通道盘和盖滑盘放入孵化器中 8 至 24 小时。为了测试Arp2/3对细胞传播的重要性，首先在控制和治疗离心管中加入5毫升的细胞培养介质。接下来，在两个较大的离心管中各加入20毫升缺乏酚红色的 dmem。派珀特要么药物CK-666，这是Arp2/3复合物的抑制剂或控制治疗，如DMSO到大小管。要开始对细胞进行药理治疗，取出在一夜之间孵育的通道盘。从所有菜肴中吸气细胞培养介质，用温暖的 PBS 洗碗。将含有 CK-666 或控制补充介质的小管放入每个通道盘中，并将相关管子的内容添加到每个通道中。给每道菜贴上正确的药物治疗标签，然后将菜肴放回孵化器中一小时后再孵化一小时，从每道菜中吸气补充药物介质。接下来，在所有菜肴中加入温暖的 PBS，以便彻底去除所有剩余的酚红色介质。加入230微升的特普辛EDTA，将菜肴放入孵化器中一分钟。从孵化器中取出试剂，然后将缺乏苯酚红的药物补充的五毫升加入指定为管 B 的管中，然后在相关菜中再加入五毫升相同介质，以淬火。将介质上下多次，以便将所有细胞从盘子中分离。将菜的所有内容转移到另一个已被指定为管子的管子中，A.In 准备适合成像的细胞稀释，将一毫升细胞从A管转移到B管中。重复每次治疗的所有稀释步骤。将所有管子放入孵化器中45分钟，使细胞从尝试性化中恢复。确保可容纳 22 乘 22 毫米盖滑的细胞磁室的所有部分在使用前都经过彻底清洁。用盖子从孵化器中取出盘子。吸气细胞培养介质，用温暖的 PBS 清洗封面滑。使用一对钳子取下盖滑，轻轻地将盖滑倒放在磁室的底部板上。接下来拿起硅胶垫片，放在盖滑的顶部。将磁室的主体连接到底部板上。加入一毫升缺乏酚红色的 dmem，与磁室的相关处理相对应。采取无绒组织，并仔细擦在主体和底部板之间的外壳，以检查任何泄漏。要完成磁室，将透明盖降低到主体上。最后用喷水的纸巾擦拭盖底，然后用70%乙醇喷洒另一块。将共聚焦显微镜的舞台顶部孵化器预热至 37 度。将磁室放在目标顶部。在获取扩散动力学之前，将焦点放在绿色荧光通道中已经两极分化的细胞上。这确保了扩散细胞在连接到盖滑时成为焦点。从从孵化器中取出的管 B 中取出磁室和移液器 500 微升的透明盖。将透明盖重新放在顶部。为了识别适合细胞扩散分析的细胞，搜索绿色光环，这些光环代表尚未附着在盖滑上的细胞或处于附着的最初阶段的细胞。获取图像并保存文件。完成细胞传播的图像采集后，首先运行兼容的 Python IDE（如 Spyder）。对于细胞传播动力学的计算分析，定位并打开相关脚本包中提供的细胞传播GUI文件。按下将打开后台分析GUI面板的运行按钮。GUI 提供分析所需的所有设置。要分析细胞区域和循环，请输入采集文件和细胞传播区域选项卡中的必要设置。必须根据单元格类型和图像采集参数进行调整。按下运行后，脚本将为所有分布的单元创建细胞循环和区域与时间图。有关基图数据，按下基图生成器和分析选项卡。此分析要求裁剪输入文件单单元格图像堆栈。在插入所有设置并按下运行后，脚本将为给定单元格创建多个基图。左边是来自DMSO和CK-666治疗的代表性细胞，使用所提供的脚本自动分割。与对照细胞所表现出的同源和高度圆形形态相反，Arp2/3抑制细胞的循环性降低。此外，自动生成的基图提供时间分辨率，对于理解突起的动态至关重要。控制细胞持续突出，几乎没有收回。相比之下，Arp2/3 抑制会破坏在整个传播过程中缩还事件增加所表明的突出的稳定性。这些视频应为您提供如何正确播种细胞、执行药理治疗以及准备细胞传播动力学量化所需的成像和计算工具的理解。此协议可与细胞球菌荧光成像和迁移测定进一步结合，以确定控制细胞突起的分子参与者。谢谢你的观察和祝你的实验好运。

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

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 ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

表面扩散和酵母染色体免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...