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
2021年11月1日
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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概述
本研究提出了一种细胞边缘凸起检测方法，用于研究细胞扩展的动态参数，包括凸起、收缩和褶皱。这种方法特别重要，因为它与细胞迁移相关，有助于识别参与细胞运动的关键蛋白和信号机制。
研究的主要组成部分
研究领域
- 细胞迁移
- 细胞运动性
- 细胞骨架动力学
背景
- 细胞边缘凸起与细胞迁移之间存在相关性。
- 该检测方法简单、经济且不需要荧光标记。
- 了解凸起可以提供细胞行为和驱动运动的机制的见解。
使用的方法
- 细胞边缘凸起检测
- 粘附在玻璃底皿上的细胞
- 用于评估凸起的动图分析
主要结果
- 凸起的平均频率为每10分钟5.1次，褶皱为2.1次。
- 动图分析显示凸起距离约为4.8微米。
- 确定了在扩展阶段选择适当细胞对于准确结果的重要性。
结论
- 该研究引入了一种可访问的检测方法来评估细胞运动动力学。
- 该方法作为在进行更复杂的迁移检测之前的初步评估工具。
常见问题
Executive Industry Relevance
Cell-edge protrusion dynamics serve as an early, predictive readout of cell motility, enabling rapid assessment of cytoskeletal regulators before committing to resource-intensive migration assays. This method supports target validation by linking molecular perturbations to functional membrane dynamics, providing mechanistic de-risking in discovery workflows. Its label-free, cost-effective design facilitates high-throughput screening of signaling pathways relevant to wound healing, immune response, and cancer metastasis.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Measures protrusion frequency, persistence, distance, and velocity to interrogate therapeutic hypotheses on actin regulators and motility pathways.
- Operational Value: Enables rapid, label-free screening of protein knockdowns or inhibitors affecting cytoskeletal dynamics.
- Predictive Value: Correlates protrusion dynamics with cell migration, supporting go/no-go decisions based on early phenotypic readouts.
Screening & Assay Development
- Scientific Value: Generates quantitative kymograph-derived metrics (protrusion count, distance, velocity) for standardized comparison across experimental conditions.
- Operational Value: Uses phase-contrast microscopy and accessible image analysis tools, reducing dependency on specialized equipment or fluorescent probes.
- Scalability: Supports multi-condition testing via radial sampling of eight membrane regions per cell, enhancing data robustness.
Translational & Preclinical Research
- Translational Continuity: Bridges molecular target modulation to functional cellular behavior in disease-relevant systems like fibroblasts or immune cells.
- Mechanistic De-risking: Clarifies whether observed phenotypic effects stem from altered protrusion dynamics rather than secondary cytotoxicity or adhesion defects.
- Biomarker Alignment: Protrusion parameters can serve as translational biomarkers when linked to in vivo motility or invasion phenotypes.
Pipeline & Workflow Integration
The assay fits within the discovery continuum from target engagement to phenotypic validation, acting as a bridge between biochemical screening and functional cell-based assays.
- Discovery Biology: Tests how genetic or pharmacological manipulations of actin regulators (e.g., Arp2/3, cofilin, Rho GTPases) impact protrusion dynamics as a proxy for motility.
- Screening: Delivers reproducible, quantitative outputs (e.g., protrusions per 10 minutes, protrusion velocity) that enable comparison across compound libraries or genetic screens.
- Analytics: Kymography provides temporal and spatial resolution of membrane dynamics, allowing extraction of kinetic parameters critical for mechanistic modeling.
- Translational Research: Supports continuity to preclinical models by validating that target hits produce consistent protrusion phenotypes in primary or disease-relevant cell lines.
- Enterprise Reuse: Establishes a standardized, low-cost platform for repeated use across projects investigating cell motility in development, inflammation, or oncology.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by directly linking molecular perturbations to quantifiable membrane dynamics.
- Operational Value: Eliminates need for fluorescent labeling and expensive imaging hardware, lowering assay barrier and increasing accessibility.
- Strategic Value: Improves capital efficiency by filtering targets early using a predictive, phenotypic anchor to cell migration.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on concordance between protrusion dynamics and migratory potential.
Implementation Considerations
- Requires expertise in live-cell imaging, kymograph generation, and manual or semi-automated protrusion analysis.
- Depends on phase-contrast microscopy and image analysis software capable of reslice and kymograph generation.
- Necessitates standardization of cell seeding density, substrate coating (fibronectin/BSA), and environmental controls (37°C, 5% CO2) for reproducibility.
- Requires training to identify appropriate spreading-phase cells and avoid confluent or contacting cells that confound protrusion measurements.
- Limited to 2D substrate-associated spreading; may not fully capture 3D invasion or confined migration phenotypes.
