Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

Nikola Lukic; Trishna Saha; Stefanie Lapetina; Michal Gendler; Gilad Lehmann; Anthony J. Koleske; Hava Gil-Henn

doi:10.3791/63157

JoVE Journal
Biology

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JoVE Journal Biology

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

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05:50 min

November 1, 2021

DOI: 10.3791/63157-v

Nikola Lukic*¹, Trishna Saha*¹, Stefanie Lapetina¹, Michal Gendler¹, Gilad Lehmann¹, Anthony J. Koleske^2,3, Hava Gil-Henn¹

¹The Azrieli Faculty of Medicine,Bar-Ilan University, ²Department of Molecular Biophysics and Biochemistry,Yale University, ³Department of Neuroscience,Yale University

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Overview

This study presents a cell-edge protrusion assay to investigate the dynamic parameters of spreading cells, including protrusions, retractions, and ruffles. This method is particularly significant as it correlates with cell migration, aiding in the identification of critical proteins and signaling mechanisms involved in cell motility.

Key Study Components

Research Area

Cell migration
Cell motility
Cytoskeletal dynamics

Background

A correlation exists between cell-edge protrusions and cell migration.
The assay is straightforward, cost-effective, and does not necessitate fluorescent labeling.
Understanding protrusions can provide insights into cellular behavior and mechanisms driving motility.

Methods Used

Cell-edge protrusion assay
Adhered cells on a glass bottom dish
Kymography analysis for assessing protrusions

Main Results

Average frequency of protrusions was 5.1 per 10 minutes, with ruffles at 2.1.
Kymography analysis revealed a protrusion distance of approximately 4.8 micrometers.
Identified the importance of choosing appropriate cells in the spreading phase for accurate results.

Conclusions

The study introduces an accessible assay to assess cell motility dynamics.
This method serves as a preliminary assessment tool before more complex migration assays are undertaken.

Frequently Asked Questions

What is the main purpose of the cell-edge protrusion assay?

The assay helps measure the dynamic parameters of cell motility, like protrusions and retractions.

Is fluorescent labeling required for this method?

No, the method is designed to be simple and cost-effective without the need for fluorescent labeling.

What are the critical metrics analyzed in this study?

Protrusions, retractions, and ruffles are analyzed to understand cell dynamics.

How does the assay contribute to biology research?

It aids in identifying proteins and signaling mechanisms involved in cell migration and motility.

What temperature conditions are required for cell incubation?

Cells should be incubated at 37 degrees Celsius during the assay.

Are there specific conditions for choosing cells for imaging?

Yes, cells must be in their spreading phase and not in contact with other cells to avoid interference.

What is the significance of kymography in this research?

Kymography is used to visualize and quantify the dynamics of the protrusions over time.

该协议旨在测量扩散细胞边缘突起的动态参数（突起，缩回，褶皱）。

细胞边缘突起测定已被证明与细胞迁移直接相关。因此，它可以用作鉴定参与细胞运动的关键蛋白质和信号传导机制的初步方法。该方法快速，简单，具有成本效益，不需要荧光标记或使用昂贵的荧光显微镜。

演示该程序的将是Michal Gendler，我实验室的学生。在玻璃底皿的中心加入两毫升一个正常的盐酸溶液，并在室温下孵育20分钟。用两毫升PBS清洗盘子三次。

在PBS中以每毫升10微克浓度稀释纤连蛋白，并将200微升稀释的溶液加入玻璃中心培养皿中。在37摄氏度下孵育一小时。准备1%BSA溶液和PBS，并通过0.2微米过滤器。

通过在预温的水浴中在70摄氏度下孵育30分钟来使溶液变性。用两毫升PBS清洗镀膜玻璃底盘三次。加入两毫升变性BSA溶液，并将培养皿在37摄氏度下孵育一小时。

用两毫升PBS清洗玻璃盘三次。实验前16~18小时，在每10厘米直径的组织培养板70.7万个细胞下达到70~80%汇合度。第二天，向组织培养板中加入两毫升胰蛋白酶溶液，孵育两到三分钟，直到细胞脱落。

加入五毫升培养基以灭活胰蛋白酶。使用血细胞计数器，在底部培养皿中将细胞和板20， 000个细胞计数在两毫升的完整培养基中。然后将培养皿与接种的细胞一起在培养箱中孵育15分钟。

打开加热装置，并在成像前一小时将其设置为37摄氏度。此外，打开二氧化碳装置，并在成像前10分钟将其设置为5%。10分钟。打开显微镜和照相机。

打开计算机并打开显微镜采集软件。在 40 倍干透镜相差上设置放大倍率。将总影片持续时间设置为 10 分钟，时间间隔为 5 秒。

孵育15分钟后，将装有粘附细胞的玻璃底培养皿放入适配器中并固定。将带有培养皿的适配器插入显微镜载物台的插槽中。取下碟盖，放置二氧化碳盖，打开二氧化碳阀门。

找到合适的成像单元。聚焦在单元格上后开始电影采集。要执行图像分析，请在图像分析软件中选择直线工具，并在每45度（包括薄片和细胞边缘）上以径向排列，使垂直于突起的8条线，每20个任意单位。

在主工具栏中，转到图像，选择堆栈，然后单击"Reslice"以生成描述细胞膜内单个点的运动记录仪图片。使用运动记录仪图像，提取并手动计算网格线标记的单元格中八个区域中每个区域的突起，缩回和褶皱的数量。这些数字表示每 10 分钟突出、收缩和褶皱的频率。

通过关节图分析确定突出物持久性，距离和速度。要确定突出距离，请绘制从突出部分的底部到突出点最高峰的垂直线。按 M 测量以像素为单位的线的长度，并确保已知像素与千分米的比率以转换以微米为单位的长度。

每10分钟手动对突起，反应和褶皱进行定量，突出物和凹陷的平均频率为5.1，褶皱的平均频率为5.1和2.1。具有代表性的运动记录仪的突出距离约为4.8微米，突出时间约为0.6分钟。表示应从分析中排除的单元格的示例。

kymography分析显示，细胞在其扩散阶段不存在，并且没有显示任何明显的膜突起。最重要的是选择正确的细胞进行成像。适当的细胞应处于扩散阶段，不应接触其他细胞，以避免细胞间通信和信号传导。

该方法可用作测试涉及细胞运动的细胞骨架动力学的初步工具，然后再决定执行更多需要细胞迁移测定的结果。

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生物学第177期细胞迁移薄片突出收缩褶皱活体成像 kymography