Summary

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Published: December 04, 2012
doi:

Summary

phagokinetic蠕动轨道法是一种方法,用于评估细胞的运动。具体而言,测定测量化学增活现象(随机细胞运动性),以定量的方式随着时间的推移。该试验利用细胞的能力,以建立一个可测量其运动轨迹胶体金涂覆盖玻片。

Abstract

单细胞和多细胞生物体细胞的活力是一个重要的生物过程。它的来源的营养物或远离不适合的条件,以及在多细胞生物组织的发展,免疫监视和伤口愈合,只是提了几个角色1,2,3的单细胞生物向运动是必不可少的。撤销这个过程可能会导致严重的神经系统,心血管疾病和免疫系统疾病,以及加剧了肿瘤的形成和传播4,5。分子,肌动蛋白聚合和受体回收已被证明发挥了重要作用,在细胞的扩展名(片状伪足),即推动向前运动的细胞6,7,8。然而,许多生物细胞迁移的问题仍然没有答案。

在人类健康和疾病的细胞运动中的核心作用突出的重要性,了解具体的MEChanisms参与了这一过程,并作出准确的方法来评估细胞的运动,特别是重要。显微镜通常是用于可视化细胞的运动。然而,细胞移动相当缓慢,耗费资源的过程,需要昂贵的相机和软件创建时间的推移定量电影运动细胞的细胞迁移的定量测量。因此,进行定量测量细胞迁移,是符合成本效益的,不费力,而且采用了常见的实验室设备的能力是一个伟大的许多研究人员的需要。

phagokinetic轨道蠕动测定法利用能力的移动单元以清除金颗粒,从它的路径建立一个可测量的磁道上的胶体金涂覆的玻璃盖玻片9,10。使用的自由软件,可以评估多个音轨,每次治疗完成统计的要求。该试验可以用来评估许多类型的细胞,如癌细胞11,12,成纤维细胞9,嗜中性粒细胞13,骨骼肌细胞14,角质形成细胞15,的滋养层16,血管内皮细胞17,和单核细胞10,18-22能动性。该协议涉及创建由柠檬酸钠,氯金酸(互惠3 +)的减少所生成的金纳米粒子(互惠°)涂覆的幻灯片。这种方法的开发由Turkevich 等人于1951年23,然后在20世纪70年代的改善Frens 。24,25。作为一个结果,该化学还原步骤中,金颗粒(10-20 nm直径)从反应混合物中沉淀,并可以应用到玻璃盖玻片,然后准备用于在细胞迁移分析9,26,27。

在一般情况下,phagokinetic轨道蠕动法是一种快速,定量和容易的细胞能动性的措施。此外,它可以用来作为一个简单的高通量的检测,使用的细胞类型,不适合时间推移的成像,以及其他用途,取决于需要的研究员。一起,定量地测量多种细胞类型的细胞运动,而不需要昂贵的显微镜和软件的能力,以及常见的实验室设备和化学品的使用,作出的phagokinetic的轨道活力测定了坚实的选择,有兴趣的科学家了解细胞蠕动。

Protocol

1。明胶涂层的盖玻片的制备放置酸洗玻璃盖玻片(15毫米直径),在无菌塑料100mm皿(晚餐)。将每道菜8-9盖玻片,并确保他们不接触对方或双方的菜.. 注:盖玻片,针和镊子必须是无菌的,以消除可能的污染的微生物,以及内毒素,这将影响细胞功能,包括蠕动。 称量明胶粉末,并重新悬浮在去离子水中的粉末,使明胶溶液的最终…

Representative Results

所示出的是示出了轨道区清零由一个单一的细胞(单核细胞从我们的实验是在图2中所示)在光学显微镜下拍摄的图像的一个例子。非运动细胞创建特征的小,椭圆形或圆形大片自己周围指示低基础水平的运动为这些未刺激的细胞( 图2A和2B)。高度运动细胞[在我们的系统中,人巨细胞病毒(HCMV)感染的细胞]与此相反,其特征在于由作为细长的轨道区?…

Discussion

轨道活力的phagokinetic在这篇文章中提出的分析是定量分析细胞迁移的一个简单而非常有效的方法。由于多种细胞类型可以分析9-17,这种方法有可能跨越多个学科的广泛使用。胶体金镀膜玻璃盖玻片的使用允许测量由移动的小区的轨道区清零。该法可衡量的效果,不同的刺激( 生长因子,纯化的ECM配体,病毒,细菌)对细胞活力。此外,缺乏监测细胞迁移的要求,进行细胞固定,…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国家卫生研究院(AI050677,HD-051998,GM103433),马尔科姆·费斯特心血管研究奖学金,和美国心脏协会博士前的奖学金(10PRE4200007)的补助支持。

Materials

Name of the reagent Company Catalog number Comments
Glass Coverslips (15 mm) Fisher Scientific 12-545-83  
Gelatin 300 Bloom Sigma-Aldrich G-1890  
Tetrachloroauric Acid Trihydrate Fisher Chemical G54-1 14.5 mM (a final working solution)
Sodium Citrate Fisher Scientific BP327-500 0.5% (a final working solution)
Paraformaldehyde Fisher Scientific O4042 3% (a final working solution)
100 mm Tissue Culture Dish Sarstedt 83.1802  
12-Well Plates Fisher Scientific 08-772-29  
24-Well Plates Fisher Scientific 07-200-84  
Techne Oven Hybridiser HB-1D LabPlanet 2040500 The standard laboratory oven will suffice
10 ml Serological Pipettes Sarstedt 86.1254.001  
Pipet-Aid Filler/Dispenser Drummond 13-681-15  
P200 Single-Channel Manual Pipette Rainin PR-200  
200 ml Barrier Tips CLP BT200  
ImageJ software http://rsb.info.nih.gov/ij/   License: Public Domain
Nikon Eclipse TE300 with a photometrics CoolSNAPfx monochrome 12-bit CCD camera Nikon   Discontinued; The most comparable specification has Nikon Eclipse Ti, but a lower end Nikon 80i will be suitable as well. Other brands also provide comparable microscopes.
      Note: The reagents and equipment listed below have been utilized by us in our various studies. Other supplies, suppliers, reagents, and equipment can be used, as long as they have similar specifications.

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Nogalski, M. T., Chan, G. C., Stevenson, E. V., Collins-McMillen, D. K., Yurochko, A. D. A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay. J. Vis. Exp. (70), e4165, doi:10.3791/4165 (2012).

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