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Apr 2019

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Single Cell Migration Assay Using Human Breast Cancer MDA-MB-231 Cell Line
利用人乳腺癌MDA-MB-231细胞系进行单细胞迁移分析   

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Abstract

Cell migration is a fundamental cellular process that plays a crucial role in many physioglogical and pathological processes such as wound healing or cancer metastasis. Many assays have been developed to examine cell migration, such as the wound healing or scratch assay, Boyden Chamber or transwell assay, and the method we will describe here, single cell migration assay. In this assay, cells are plated sparsely on a collagen coated plate and live cell imaging is performed over a period of 2 h at 1 frame per minute. After imaging is completed, cells are tracked manually using ImageJ by tracking movement of the centroid of the cell. These data points are then exported and overall distance travelled from frame to frame is determined and divided by total time imaged to determine speed of the cell. This method provides a quick way to examine effect of cellular manipulation on cell migration before proceeding to perform more complex assays.

Keywords: Single cell migration (单细胞迁移), Cell tracking (细胞示踪), 2D migration (2D迁移), Random migration (随机迁移), Live cell imaging (活细胞成像)

Background

Cell migration plays an important role in both physiological and pathological processes ranging from embryonic development to angiogenesis and tumor metastasis (Le Clainche and Carlier, 2008). Cell motility is a highly orchestrated event that can be summarized as a cycle of four basic steps: i) membrane protrusion driven by actin polymerization, ii) stabilization of protrusion through integrin-mediated cell-matrix adhesion, iii) cell-body translocation driven by actomyosin contractile force, and finally, iv) rear release as a result of the mechanical action of contractile force and/or proteolysis of cell-matrix adhesion components (Sheetz et al., 1999; Ridley et al., 2003; Panetti et al., 2004; Stradal and Scita, 2006; Tomasevic et al., 2007; Le Clainche and Carlier, 2008). These processes involve dynamic remodeling of actin cytoskeleton which is dependent on de novo synthesis as well as regulation of important structural and regulatory components of actin cytoskeletal system.

Several methods have been developed to examine cell migration and can be separated into 2D and 3D assays. 2D migration assay have their advantages as they are typically easier and quicker to perform, however, lack the physiological representation that 3D assays may provide. We will provide a brief description of some of the other methods for cell migration. The scratch assay or wound healing assay is a commonly used technique to evaluate directed cell migration (Cory, 2011). In this assay, cells are plated to form a confluent monolayer and a stratch is introduced in the monolayer. The cell-free zone is then monitored as adjacent cells migrate to close the gap. While simple to perform, cell proliferation may bias the readout of this assay as that can influence scratch closure. In addition, cells in this assay are forced to move in one direction. The Boyden Chamber assay or transwell assay is another technique used to evaluate cell migration (Falasca et al., 2011). In this assay, cells are placed on one side of a porous membrane and allowed to migrate through the pores to the other side. An advantage of this assay is it allows for chemotaxis and works for both adherent and non-adherent cells. As with the scratch assay, migration in this assay is defined. Some disadvantages of this assay include difficulty to visualize cells and morphology due to transitive state of cells while migrating through pores.

In this protocol, we will describe the use of single cell migration assay to examine MDA-MB-231 cell migration. The key difference in this protocol is that we examine random cell migration rather than directed migration. This allows us to examine cell migration in a manner that is less influenced by cell-cell contact and its innate ability for directional persistence, i.e., ability to migrate randomly in a single direction. While obviously not a true physiological representation of in vivo cell migration, this assay allows us to quickly analyze effect of cellular manipulation on its ability to migrate. While this assay uses MDA-MB-231, this protocol can be performed with any migratory cell line. We have performed this assay using multiple cell lines with minor modifications (media used and amount of cells plated).

Materials and Reagents

  1. 24-well cell culture plate (Corning, Costar, catalog number: 9761146 )
  2. 100 mm cell culture plate (Corning, catalog number: 08-772-22 )
  3. 15 ml conical centrifuge tubes (Falcon, catalog number: 14-959-49D )
  4. 100% CO2 Tank
  5. DMEM (Lonza, BioWhittaker, catalog number: BW12-604F )
  6. Heat-inactivated FBS (Corning, catalog number: 35011CV )
  7. 100x Antibiotic-Antimycotic (Gibco, catalog number: 15240-062 )
    Note: This is optional. We use this as extra assurance to prevent bacterial and fungal contamination in our cell culture. For some cell lines, this can hinder cell growth.
  8. 100x Sodium pyruvate (Gibco, catalog number: 11360-070 )
  9. Collagen I, 3 mg/ml (Gibco, catalog number: A1048301 )
  10. Trypsin (Lonza, Trypsin-Versene, catalog number: BW17161E )
  11. PBS (Lonza, BioWhittaker, catalog number: BW17516F )
  12. MDA-MB-231 (ATCC, catalog number: CRM-HTB-26 )
  13. DMEM S+ media (see Recipes)
  14. Collagen (10 μg/ml) (see Recipes)

Equipment

  1. Hemocytometer
  2. Live-cell imaging environmental chamber (Tokai Hit live-cell environmental chamber)
  3. Centrifuge (unrefrigerated) for 15 ml tubes (Sorvall, Legend RT, SO-LEGRT)
  4. Cell incubator set to 37 °C and 5% CO2
  5. Waterbath or drybath set to 37 °C (Boekel Scientific, Small Water Bath, 290100)
  6. Microscope with automatic stage control (Olympus IX71 Inverted Phase with Prior Scientific motorized stage) (Figure 1)
  7. Camera for microscope (Hamamatsu)
  8. 10x objective (Olympus)


    Figure 1. Example of a complete setup for cell tracking using Olympus IX71 inverted microscope, Prior mechanized stage, and Tokai Hit environmental chamber

Software

  1. Software compatible with microscope for time-lapse image aquisition (Olympus, cellSens)
  2. Fiji ImageJ (https://fiji.sc/)
  3. Microsoft Excel

Procedure

  1. Cell culture
    1. Warm up DMEM S+ media to 37 °C.
    2. Thaw MDA-MB-231 cells to 37 °C.
    3. Transfer 1 ml of thawed MDA-MB-231 cells to 3 ml of DMEM S+ media and centrifuge (with appropriate balance) for 3 min at 300 x g.
    4. Aspirate supernatant and resuspend cells in MDA-MB-231 cells in 10 ml of DMEM S+ and plate on 10 cm dish.
    5. Incubate cells at 37 °C and 5% CO2 until 70% confluency (2-3 days).

  2. Single cell migration assay
    1. Warm up DMEM S+ media, trypsin, and PBS to 37 °C.
    2. Coat 24-well plate with 10 μg/ml Collagen-I solution (100 μl) for 1 h at room temperature and wash with PBS (leave PBS in well to prevent drying).
    3. Aspirate media from MDA-MB-231 cell culture dish.
    4. Wash cells with PBS and aspirate PBS.
    5. Add 1 ml of trypsin and incubate in 37 °C and 5% CO2 for 5 min.
    6. Pick up trypsinized cells with 3 ml of DMEM S+ and centrifuge for 3 min with appropriate balance at 300 x g.
    7. Aspirate supernatant and resuspend cells in 4 ml of DMEM S+.
    8. Take 10 μl of cell suspension and pipette into hemocytometer.
    9. Count cells.
    10. Aspirate PBS off collagen coated 24-well plate.
    11. Resuspend cell suspension again by inverting tube a few times or pipetting.
    12. Plate 20,000 cells into 24-well and allow to attach overnight at 37 °C and 5% CO2.
    13. Allow live-cell imaging environmental chamber warm up on microscope stage with empty 24-well plate for at least 1 h to equilibrate microscope temperature.
    14. Replace empty plate with cell plated 24-well and allow to equilibrate for 1 h.
    15. Select multiple fields with 5-10 single cells (not touching other cells) and set time-lapse imaging for 2 h at 1 frame per minute using phase-contrast.
    16. Initiate time-lapse imaging and save files.

Data analysis

  1. If fields were saved as individual images (i.e., 1 image per field per time point), import image sequence on Fiji to build a montage of all images from one field. If fields were saved as an individual file (i.e., 1 file per field), open directly with Fiji.
  2. Play through the movie and identify cells that do not touch other cells or migrate off the field of view (try to identify 5-10 cells or more per field is possible). See Video 1 for example field of migrating cells.

    Video 1. Example video of cell migration from Figure 2

  3. Once candidate cells have been determined, open Manual Tracking plugin on Fiji (Figure 2A, a window should open).
  4. Click “add track” (Figure 2B).
  5. Begin clicking on the centroid of cell (center of cell) and continue tracking until all 120 frames have been tracked (Figure 2C). Note that data will be added to a new window (Figure 2D).
  6. Click “end track” and close manual tracking window (Figure 2E).


    Figure 2. Workflow for manual tracking using Fiji. A. Select Manual Tracking under Plugins. B. Click Add Track. C. Begin tracking a cell (continuously click on centroid of cell, for example, as illustrated by white arrow. Note that cells that are clumped together like those in the middle of this panel should not be used for tracking. D. Tracking information will be generated by ImageJ. E. Click End Track when tracking is done.

  7. Save results window which contains X, Y coordinates of the cell centroid per frame.
  8. Repeat Steps 3-7 until all cells have been quantified and proceed to next field.
  9. Once all cells and fields have been quantified, open Excel and paste X,Y coordinates (Figure 3A).
  10. Determine distance travelled from point to point by using the distance formula below where X1 and Y1 are the current track location and X2 and Y2 are the next track location. Find d for each tracked point (Figure 3B, there will be 121 tracked points, since there is no previous point to the first tracked spot, you will end up with 120 calculations for d).




    Figure 3. Example of data analysis using Excel. A. Copy over X and Y coordinates from cell tracking into Excel and line up side-by-side for each cell tracked. B. In separate section, use the distance formula to calculate distance travelled by cell from Point 1 to Point 2, Point 2 to Point 3, Point 3 to Point 4, and so on. Example of Excel function used is highlighted.

  11. Add up all distance travelled per cell and divide by 120 (this is the duration of the movie in min and also the number of frames per field).
  12. Repeat Steps 10 and 11 for all cells.
  13. Summarize cell speed as desired and perform appropriate statistical analysis (t-test for 2 groups, ANOVA with post hoc for > 2 groups).
  14. Plot summarized information using preferred graph (i.e., bar graph, box-whisker, scatter plot). An example of a box-whisker plot is shown in Figure 4.


    Figure 4. Example of speed results plotted as a box and whiskers chart using Excel. G1, G2, G3 are generic group names. The blue star represents the mean and the green bar represents the median of the data.

Recipes

  1. DMEM S+ media
    500 ml DMEM
    50 ml FBS
    5 ml 100x Antibiotic-Antimycotic
    5 ml 100x Sodium Pyruvate
  2. Collagen (10 μg/ml)
    10 ml DMEM
    166.6 μl Collagen I 3 mg/ml

Acknowledgments

This work was supported by a grant from the National Institute of Health (2R01CA108607) to PR. David Gau was supported by a National Science Foundation pre-doctoral fellowship (2012139050) and an NIH Cardiovascular Bioengineering pre-doctoral training grant (2T32HL076124 to SG).

Competing interests

The authors have no competing interests to report.

References

  1. Cory, G. (2011). Scratch-wound assay. Methods Mol Biol 769: 25-30.
  2. Falasca, M., Raimondi, C. and Maffucci, T. (2011). Boyden chamber. Methods Mol Biol 769: 87-95.
  3. Le Clainche, C. and Carlier, M. F. (2008). Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev 88(2): 489-513.
  4. Panetti, T. S., Hannah, D. F., Avraamides, C., Gaughan, J. P., Marcinkiewicz, C., Huttenlocher, A. and Mosher, D. F. (2004). Extracellular matrix molecules regulate endothelial cell migration stimulated by lysophosphatidic acid. J Thromb Haemost 2(9): 1645-1656.
  5. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz, A. R. (2003). Cell migration: integrating signals from front to back. Science 302(5651): 1704-1709.
  6. Sheetz, M. P., Felsenfeld, D., Galbraith, C. G. and Choquet, D. (1999). Cell migration as a five-step cycle. Biochem Soc Symp 65: 233-243.
  7. Stradal, T. E. and Scita, G. (2006). Protein complexes regulating Arp2/3-mediated actin assembly. Curr Opin Cell Biol 18(1): 4-10.
  8. Tomasevic, N., Jia, Z., Russell, A., Fujii, T., Hartman, J. J., Clancy, S., Wang, M., Beraud, C., Wood, K. W. and Sakowicz, R. (2007). Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2. Biochemistry 46(11): 3494-3502.

简介

[摘要 ] 细胞迁移是一个基本的细胞过程,在许多生理和病理过程中(如伤口愈合或癌症转移)起着至关重要的作用,已经开发了许多检测细胞迁移的方法,例如伤口愈合或刮擦实验,Boyden Chamber或Transwell 分析,我们将在此介绍的方法是单细胞迁移分析。在此分析中,将细胞稀疏地铺在胶原蛋白包被的板上,并在2小时内进行活细胞成像 成像完成后,通过跟踪细胞质心的运动使用ImageJ手动跟踪细胞,然后导出这些数据点,确定帧与帧之间的总距离并除以成像的总时间确定细胞的速度。此方法提供了一种在进行更复杂的测定之前检查细胞操作对细胞迁移影响的快速方法。

[背景 ] 细胞迁移在从胚胎发育到血管生成和肿瘤转移的生理和病理过程中均起着重要作用(Le Clainche 和Carlier ,2008)。细胞运动是高度精心策划的事件,可以概括为四个基本步骤的循环:我)膜伸出驱动下肌动蛋白聚合,二)稳定突出通过整合素介导的细胞-基质粘附,III)细胞体易位驱动通过肌动球蛋白收缩力,最后,IV)后发布结果的Mechan 的iCal力和/或细胞基质粘附组分的蛋白水解作用(Sheetz 等,1999 ; Ridley 等,2003; Panetti 等,2004; Stradal and Scita ,2006; Tomasevic 等,2007; Le Clainche and Carlier ,2008)。这些过程涉及肌动蛋白细胞骨架的动态重塑,它依赖于从头合成以及肌动蛋白细胞重要结构和调节成分的调节。骨骼系统。

几种方法已经发展到检查细胞迁移和可被分为2D和3D试验。2D迁移分析有其优势,因为他们通常更容易和更快地执行,但是缺乏生理表达形式。它的3D试验提供可能。我们会提供简要介绍其他一些用于细胞迁移的方法。刮擦测定或伤口愈合测定是评估定向细胞迁移的常用技术(Cory,2011)。在该测定中,将细胞铺板以形成汇合的单层和将Stratch 引入单层中,然后在相邻细胞迁移以闭合间隙时监测无细胞区。在执行过程中,细胞增殖可能会影响该测定的读数,因为这可能会影响刮痕闭合。这种测定法被迫朝一个方向移动。博伊登室测定法或Transwell 测定法是另一种用于评估细胞迁移的技术(Falasca 等,2011)。这种测定法的一个优点是可以进行趋化作用,并且对粘附细胞和非粘附细胞都有效。与刮擦测定法一样,迁移进入的过程被置于多孔膜的一侧,并允许通过孔迁移至另一侧。定义了该测定法。该测定法的一些缺点包括由于细胞在通过孔迁移时的传递状态而难以可视化细胞和形态。

在该协议中,我们将描述使用单细胞迁移测定来检查MDA-MB-231细胞迁移的方法,该协议的主要区别在于我们检查随机细胞迁移而不是定向迁移,这使我们能够检查一种不受细胞间接触及其固有的定向持久能力(即在单个方向上随机迁移的能力)的影响较小的方式。虽然该方法并非真正的体内细胞迁移的生理学表现,但它使我们能够快速分析尽管此测定法使用MDA-MB-231,但该方案可在任何迁移细胞系中进行。我们已经使用多个细胞系进行了此测定法,并进行了较小的修改(使用的培养基和细胞数量)。镀)。

关键字:单细胞迁移, 细胞示踪, 2D迁移, 随机迁移, 活细胞成像

材料和试剂


 


24 - 孔细胞培养板(Corning公司,Costar公司,目录号:9761146)
100 mm细胞培养板(Corning,目录号:08-772-22)
15 ml 锥形离心管(Falcon,目录号:14-959-49D)
100%CO 2 储罐
DMEM(Lonza ,BioWhittaker ,目录号:BW12-604F)
热灭活的FBS(Corning,目录号:35011CV)
100x抗生素-抗真菌药(Gibco,目录号:15240-062)
注意:Thi s是可选的,我们以此作为防止细胞培养中细菌和真菌污染的额外保证,对于某些细胞系,这可能会阻碍细胞生长。


100x丙酮酸钠(Gibco,目录号:11360-070)
I型胶原蛋白,3 mg / ml (Gibco,目录号:A1048301)
胰蛋白酶(Lonza,Trypsin- Versene ,目录号:BW17161E)
PBS (Lonza ,BioWhittaker ,目录号:BW17516F)
MDA-MB-231(ATCC,目录号:CRM-HTB-26)
DMEM S + 介质(请参阅食谱)
胶原蛋白(10μg / ml)(请参阅食谱)
 


配套设备


 


血细胞计数器
活细胞成像环境室(Tokai Hit活细胞环境室)
离心(未冷藏)的15毫升试管(Sorvall ,Legend RT,SO-LEGRT)
细胞培养箱设置为37 °C和5%CO 2
水浴或干浴温度设置为37°C(Boekel Scientific,小型水浴,290100)
具有自动镜台控制的显微镜(具有Prior Scientific电动雄鹿的Olympus IX71反相)(图1)
显微镜用相机(滨松)
10倍物镜(奥林巴斯)
 


D:\重新格式化\ 2020-2-7 \ 1902750--1320 David Gau 782084 \图jpg \图4.jpg


图1. 使用Olympus IX71倒置显微镜,先前的机械化工作台和Tokai Hit环境室进行细胞跟踪的完整设置示例


 


软体类


 


与显微镜兼容的用于延时图像采集的软件(奥林巴斯,cellSens)
斐济ImageJ(https://fiji.sc/)
微软Excel
 


程序


 


细胞培养
将DMEM S +介质预热到37°C。
将MDA-MB-231细胞解冻至37°C。
将1 ml解冻的MDA-MB-231细胞转移至3 ml DMEM S +培养基中,并以300 xg 离心(适当平衡)3分钟。
吸出10 ml DMEM S +中的MDA-MB-231细胞中的鸡并重悬细胞,然后在10 cm的培养皿中平板接种。
在37°C和5%CO 2 下孵育细胞,直到70%融合(2-3天)。
 


单细胞迁移测定
将DMEM S +培养基,胰蛋白酶和PBS预热至37°C。
24大衣-好板带10MYU ģ / ml的I型胶原溶液(100 Myueru )1个小时在室温和洗涤用PBS(PBS假中公以防止干燥)。
从MDA -MB -231细胞培养皿中吸出培养基。
用PBS和吸出PBS洗涤细胞。
加入1 ml的胰蛋白酶,并在37°C和5%CO 2中孵育5分钟。
用3 ml DMEM S + 提取经胰蛋白酶处理的细胞,并以300 x g的适当平衡离心3分钟。
吸出民意调查和重悬细胞在4毫升的DMEM S +。
10取MYU 大号细胞悬浮液,液吸入血球。
计数细胞。
PBS关胶原吸涂24 - 孔板。
通过颠倒几次试管或移液再次重悬细胞悬液。
将20,000个细胞置于24孔中,并在37°C和5%CO 2 下附着过夜。
活细胞成像允许环境商会热身在显微镜台上空24 - 孔板至少1 小时,下平衡显微镜温度。
空盘替换为细胞镀24 - 好吧,让平衡1 H.
用5-10 s的ingle单元(不触摸其他单元)选择多个场,并使用相衬将延时成像设置为每分钟1帧2 h。
启动延时成像并保存文件。
 


资料分析


 


如果将字段另存为单个图像(即每个时间点每个字段1个图像),请在斐济上导入图像序列以构建一个字段中所有图像的蒙太奇。如果将字段另存为单个文件(即每个字段1个文件) ),直接在斐济打开。
播放电影并确定不与其他单元格接触或移出视场的单元格(尝试识别每个视场5-10个或更多单元格)。有关移动单元格的示例,请参见视频1。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-4-20 \ 3586--1902750--1320 David Gau 782084 \ video 1.jpg


视频1.图2中的细胞迁移示例视频


 


候选细胞具有一旦被确定,打开手动跟踪插件斐济(图茜2 A,A窗口应该开)。
点击“添加轨道”(图2 B)。
开始单击单元格的质心(单元格的中心)并继续跟踪,直到已跟踪所有120帧(图2 C)。请注意,数据将添加到新窗口中(图2 D)。
单击“结束跟踪”,然后关闭手动跟踪窗口(图2 E)。
 


D:\重新格式化\ 2020-2-7 \ 1902750--1320 David Gau 782084 \图jpg \ 1.jpg


FIGUR ê 2 。工作流程手动跟踪使用斐济。一。选择手动跟踪在插件; B 。点击添加轨道。Ç 。开始跟踪小区(连续按质心细胞,例如,如图所示的白色箭头。请注意,这是蝌蚪细胞在一起,就像那些在中间这个小组不应该被用于跟踪。d 。跟踪信息将产生通过ImageJ的。ê 。点击结束轨迹跟踪时完成。


 


保存结果窗口,其中每帧包含单元质心的X,Y坐标。
重复步骤3-7,直到所有单元格都已量化,然后进入下一个字段。
所有的细胞,一旦领域都得到了量化,打开Excel和粘贴X ,Y 坐标(图3 A)。
确定距离专程从点到点通过使用距离公式下面其中X 1 和Y 1 是当前轨道位置和X 2 和Y 2 是下轨位置。求d对于每个跟踪点(图茜3 B,将有是121个跟踪点,因为没有第一个跟踪点的先前点,您将最终得到d)的120个计算。
 






 


D:\重新格式化\ 2020-2-7 \ 1902750--1320 David Gau 782084 \图jpg \ 2.jpg


图3 ,使用Excel例数据分析的一个。复制在X和Y坐标从细胞跟踪到Excel中,阵容并排侧履带; B每一个细胞。在单独的一个部分,使用距离公式来计算距离旅行按单元格从点1到点2,点2到点3,点3到点4,依此类推。突出显示所用Excel函数的示例。


 


将每个像元所经过的所有距离相加并除以120(这是影片的持续时间(以分钟为单位,也是每个场的帧数))。
对所有单元格重复步骤10和11。
总结所需的细胞速度并进行适当的统计分析(t 检验2组,ANOVA进行事后分析> 2组)。
使用首选图表(即条形图,箱须图,散点图)绘制汇总信息。图4中显示了箱须图的示例。
 


D:\重新格式化\ 2020-2-7 \ 1902750--1320 David Gau 782084 \图jpg \图3.jpg


Figu 重4 。例子的速度结果绘制成盒和胡须图表使用Excel中。G1,G2,G3是通用的组名。蓝星代表均值和绿条表示的位数的数据。


 


菜谱


 


DMEM S +媒体
500毫升DMEM


50毫升FBS


5毫升100 x抗生素-抗真菌药


5毫升100x丙酮酸钠


胶原(10 MYU ģ / ml)的
10毫升DMEM


166.6 MYU 大号胶原I 3 毫克/毫升


 


致谢


 


这项工作得到了美国国立卫生研究院(PR)的资助(2R01CA108607)。David Gau得到了美国国家科学基金会的博士前研究金(2012139050)和NIH心血管生物工程博士前研究资助(SG的2T32HL076124)的支持。 。


 


 


竞争利益


 


作者没有竞争利益要报告。


 


参考文献


 


Cory,G.(2011)。刮伤试验。方法,分子生物学769:25-30。
Falasca ,M.,莱蒙迪,C。和Maffucci ,T。(2011)。Boyden小室。方法分子生物学杂志769:87-95。
勒Clainche ,C。和CARLIER ,MF(2008)。迁移调控肌动蛋白组件相关联随着突起和粘附在细胞。生理学版本88(2):489-513。
Panetti ,TS,Hannah,DF,Avraamides,C.,Gaughan ,JP,Marcinkiewicz ,C.,Huttenlocher ,A。和Mosher,DF(2004)。细胞外基质分子调节溶血磷脂酸刺激的内皮细胞迁移, J Thromb Haemost 2 (9):1645-1656。
雷利,AJ,施瓦茨,MA,伯里奇,K.,Firtel ,RA,金斯堡,MH&LT ;, Borisy ,G.,帕森斯,JT和Horwitz,AR(2003)。细胞迁移:.积分信号由前向后科学302( 5651):1704-1709。
Sheetz,中号。P 。,Felsenfeld ,d 。,加尔布雷思,Ç 。ģ 。而且的Choquet ,D.(1999)。迁移作为细胞五步。周期生物化学志SYMP 65:233-243。
Stradal ,TE和Scita ,G.(2006)。调节 Arp2 / 3介导的肌动蛋白装配的蛋白质复合物。Curr Opin Cell Biol 18(1):4-10。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gau, D. M. and Roy, P. (2020). Single Cell Migration Assay Using Human Breast Cancer MDA-MB-231 Cell Line. Bio-protocol 10(8): e3586. DOI: 10.21769/BioProtoc.3586.
  2. Gau, D., Veon, W., Shroff, S. G. and Roy, P. (2019). The VASP-profilin1 (Pfn1) interaction is critical for efficient cell migration and is regulated by cell-substrate adhesion in a PKA-dependent manner. J Biol Chem 294(17): 6972-6985.
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