随机使用时移视频显微镜细胞迁移的定量分析
Summary
这种方法允许在不同的细胞迁移参数，如速度，位移和速度的实时性和定量测量细胞的监测。实时方式与传统方法不同的是，这不是基于端点的定量迁移测量;相反，它允许不同的参数，不断监测和计算。
Abstract
细胞迁移是一个动态的过程，这是非常重要的胚胎发育，组织修复，免疫系统的功能，与肿瘤的侵袭1，2。在定向迁移，细胞外趋化信号的响应，或响应内在线索3的基本动力机械提供快速移动。随机迁移发生时，细胞具有低固有的方向性，使细胞探索当地环境。
细胞迁移是一个复杂的过程，在最初的反应细胞，经过极化和中迁移的方向延伸的突起。传统的方法来衡量，如Boyden小室迁移实验迁移是一个简单的方法来衡量在体外 ，从而使一个终点的结果作为衡量迁移的趋化。不过，这种做法既不允许个体迁移参数的测量，也不允许到visualization的细胞在迁移过程中经历的形态学变化。
在这里，我们提出了一种方法，使我们能够监测细胞迁移，在实际使用视频时间 – 时间的推移显微镜。由于细胞迁移和侵袭癌的标志，这种方法适用于研究癌症细胞迁移和体外侵袭。随机迁移的血小板已考虑为血小板功能的4参数之一，因此这种方法也可以是血小板功能的研究很有帮助。此法的优点是快速，可靠，重现性好，不需要手机号码的优化。为了维持细胞生理适当的条件下，在显微镜配备CO 2供应和温控器。细胞的运动进行监测使用安装在显微镜定期相机拍照。细胞迁移可以通过测量平均时速和计算verage位移，这是由Slidebook软件计算。
Protocol
1。胶原涂层板的制备胶原蛋白稀释到终浓度为10微克/毫升的Opti-MEM媒体。 大衣胶原的6孔板，从第1步，每孔加入1.5毫升。孵育过夜在4°C 翌日，1X磷酸的2倍缓冲液（PBS）洗板，将它们存储在PBS。 2。在6孔板细胞的制备及幼苗 注：用温水媒体和PBS，以确保细胞运动的最佳条件成长的MDA-MB-231（浸润性乳腺癌细胞?…
Discussion
实时随机的移民法能够准确，灵敏的细胞迁移的参数，如速度和位移分析。此方法不仅限于结束点的测量值，因此它是更多的量化。由于细胞在正常培养液血清培养基监测，它减少了有害影响较长的潜伏期在无血清媒体。它已被证明的格式和基质8，因此，这种方法可以用来研究不同基质对移民的影响，打破了细胞接触依赖的细胞迁移。
的关键步骤之一，包括适当的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢阿曼达Struckhoff与实验的初始帮助。这项工作是从5RO1CA115706 NIH的拨款支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| DMEM | Thermo Scientific | SH30243.01 | |
| FBS | Gemini Bio- Products | 100-106 | |
| Opti-Mem | Invitrogen | 31985-070 | |
| Collagen | BD | 354231 | |
| Microscope with Live Cell chamber & Automated XY stage controller | Olympus | Olympus 1X81 | |
| Environmental control chamber | Neue | Neue Live Cell Chamber | Ours has a custom built rectangular glass plate top for appropriate optics through the top of the chamber. The rectangular shape accommodates multi-well plates used in many of our experiments. |
| Automated XY stage | Prior | Prior Proscan | This allows precise return to multiply selected XY positions during time lapse microscopy. |
| Camera | Hamamatsu EM camera C9100 | ||
| Software | Typically purchased through microscope vendor such as Olympus | Slide Book 5 |
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Tags
Quantitative AnalysisRandom MigrationCellsTime-lapse Video MicroscopyCell MigrationEmbryonic DevelopmentImmune System FunctionTumor InvasionChemotactic SignalIntrinsic CuesMotility MachineryLocal EnvironmentPolarizationProtrusionsBoyden Chamber Migration AssayChemotaxisEnd Point ResultIndividual Migration ParametersMorphological ChangesCancer Cell MigrationCancer Cell InvasionPlateletsPlatelet Function
Citar este artigo
Jain, P., Worthylake, R. A., Alahari, S. K. Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy. J. Vis. Exp. (63), e3585, doi:10.3791/3585 (2012).