创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移
Summary
为组件的粘接剂和可溶性的梯度在显微镜室，用于活细胞迁移研究的方法，进行说明。该工程环境结合溶液梯度的防污表面和粘接剂的轨道，并因此允许一个确定指导线索的相对重要性。
Abstract
细胞可以感知并移向较高浓度的粘合剂线索，如糖蛋白如生长因子的细胞外基质和可溶性线索。在这里，我们列出了反对梯度的粘合剂和可溶性线索，微流控室，这是兼容与活细胞成像的方法来创建。聚-L-赖氨酸和聚乙二醇（PLL-PEG）的共聚物，以钝化玻璃的盖玻片和防止生物分子和细胞的非特异性吸附。往下，微接触印刷或蘸笔光刻被用来创建轨道的链霉抗生物素蛋白上的钝化表面的生物素化的肽，精氨酸 – 甘氨酸 – 天门冬氨酸（RGD）作为粘接剂提示作为锚固点。一种微流体装置被放置到改性的表面上，并用于创建的梯度粘合剂线索（100％到0％的RGD RGD）上的抗生蛋白链菌素的轨道。最后，相同的微流体装置是用于创建的单核细胞趋化自吸的梯度h为牛胎儿血清（FBS），在相反的方向上的梯度的粘合剂线索作为可溶性提示。
Introduction
导演细胞迁移是许多细胞的基本属性，是许多正常的生理过程，包括胚胎发育，抗感染，促进伤口愈合的一个重要方面。此外，细胞迁移也起到了突出的作用，在许多疾病，如血管疾病，肿瘤细胞的转移和慢性炎症1,2。虽然传统的状态的细胞迁移-极化，凸出延伸，形成粘连，力产生收缩和后收缩-被普遍接受的3,4，阐明的时空信号集成协调机制，已是更具挑战性。
的胞外基质（ECM）作为细胞粘附的基板，并通过固有的化学和物理6多样性，提供用于细胞导航7,8的方向提示。除了 这些粘合剂线索9，可溶性因子10-12 </ SUP>，如趋化因子和生长因子，可诱导细胞迁移通过定向趋化因子受体和其下游motogenic的信令。目前，参与和信令通过粘附受体（ 即整合），或如趋化因子受体，受体酪氨酸激酶（RTK），不知道是否是显性的。也不知道是否是细胞类型特异性受体系统的层次结构。
活细胞显微镜提供了丰富的信息，是不是可以批量检测，可以结合微流体装置，产生梯度的固定13,14和可溶性线索15 16。这里描述的方法采用了一系列的简单和既定一并用市售的微流体腔室，以创建一个成像检测细胞的迁移，可以很容易地在细胞生物学实验室（ 图1）实施表面改性步骤。组装的微流体装置具有与玻璃17的光学品质的梯度的扩散的分子是稳定的，至少16小时。该系统可用于落射荧光和先进的显微镜技术。不像其他的趋化性的设置18，这个系统是适合用于记录缓慢迁移，贴壁细胞。更重要的是，该系统是模块化的，很容易使引进替代的粘合剂或可溶性迁移暗示的各种细胞类型和检查。
Protocol
1。 PLL-PEG-生物素玻片上与钝化这一步的目的是钝化的表面，从而使细胞的粘附和迁移到特定区域的盖玻片上所建立的（步骤2-3A）microcontract印刷或的蘸水笔平版印刷（步骤3b）。对于钝化，一个共同的聚合物的聚-L-赖氨酸（PLL）和聚乙二醇（PEG）中，使用20％的PEG分子被接枝到生物素（PLL-PEG-生物素）。 要清洁的玻璃盖片（18毫米×18毫米），浸没在100％乙醇中的齿条，…
Representative Results
为了了解细胞整合洄游信号25，我们已经开发了一种方法的图像细胞用荧光显微镜观察，与竞争的粘接剂和可溶性的梯度（ 图1）的环境中迁移。胶粘剂的轨道含有荧光链霉亲和素和生物素标记的RGD创建的微接触印刷的曲目和蘸笔光刻（ 图2）。成功的微接触印刷粘合剂轨道和防污区域可以清楚地加以区分（ 图2b）的磁道之间的荧光强度表示的线剖面?…
Discussion
在这个协议中，我们使用市售的微流体装置（粘性滑动的趋化三维 Ibidi）的化学表面改性及对细胞迁移的趋化因子梯度的影响进行研究。因为梯度设立的沿该通道的长度的扩散，这种微流体设置不需要流。这是很重要的，因为流量差异影响慢慢迁移的细胞，如成纤维细胞，形成稳定的粘着斑和快速迁移细胞最喜欢的白细胞，形成协调配合物与突触小26。从层流剪应力可能影响稳定?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者承认资金从澳大利亚研究理事会和国家健康与医学研究委员会澳大利亚也想感谢 澳大利亚国家的制造工厂主的微接触印刷的SU-8。 SHN支持的马来西亚高等教育和马来西亚理科大学部。
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| Coverglass staining outfits | Thomas Scientific | 8542 E40 | Coverslip rack |
| Oven | Binder | ED 53 series | |
| Silicon wafer | Silicon Quest | 708-007 | Boron doped <100> wafer, 4″ diameter, 500 μm, single side polished |
| GM1070 SU-8 photoresist | Gersteltec Sarl | ||
| SU-8 developer | Gersteltec Sarl | ||
| Sylgard 184 curing agent | Dow Corning | ||
| Sylgard 184 elastomer prepolymer | Dow Corning | ||
| PLL-PEG-biotin (20%) | SuSos AG | PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%) | 1 mg/ml in PBS |
| Fluorescein | Sigma | 46955 | 1 mM in PBS |
| Streptavidin-AlexaFluor350 | Invitrogen | S-11249 | 1 mg/ml in PBS |
| Biotin-4-fluorescein | Invitrogen | B-1370 | 0.03 μg/μl in PBS |
| Biotin-RGD | GenScript | SC1208 | 0.03 mg/ml in PBS |
| Syto 64 Red | Invitrogen | S-11346 | 1 μM in PBS |
| Sticky slide chemotaxis 3D | Ibidi | 80328 | |
| 200 μl Greiner yellow bevelled tip | Greiner Bio-One | 739261 | |
| Vaseline | Sigma | 16415 | |
| Paraffin wax, mp 55-57 °C | Sigma | 327204 | |
| Nano eNabler 10 μm cantilever | BioForce | SPT-S-C-10s | |
| Image J software | National Institute of Health | rsbweb.nih.gov/ij/download.html | |
| Manual Tracking plugin | Fabrice Cordelières | rsb.info.nih.gov/ij/plugins/track/track.html | |
| Chemotaxis and Migration Tool | Ibidi GmbH | www.ibidi.com/applications/ap_chemotaxis.html |
References
- Pulsipher, A., Yousaf, M. N. Surface Chemistry and Cell Biological Tools for the Analysis of Cell Adhesion and Migration. Chem. BioChem. 11, 745-753 (2010).
- Hall, A. The cytoskeleton and cancer. Cancer Metastasis Reviews. 28, 5-14 (2009).
- Parsons, J. T., Horwitz, A. R., Schwartz, M. A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 11, 633-643 (2010).
- Geiger, B., Spatz, J. P., Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21-33 (2009).
- Roussos, E. T., Condeelis, J. S., Patsialou, A. Chemotaxis in cancer. Nature Reviews Cancer. 11, 573-587 (2011).
- Petrie, R. J., Doyle, A. D., Yamada, K. M. Random versus directionally persistent cell migration. Nat. Rev. Mol. Cell Biol. 10, 538-549 (2009).
- Geiger, B., Yamada, K. M. Molecular architecture and function of matrix adhesions. Cold Spring Harbor Perspectives in Biology. 3, (2011).
- Ngalim, S. H., Magenau, A., Saux, G. L. e., Gooding, J. J., Gaus, K. How do cells make decisions: engineering micro- and nanoenvironments for cell migration. Journal of Oncology. 2010, 363106 (2010).
- Roy, D. C., Wilke-Mounts, S. J., Hocking, D. C. Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate. Biomaterials. 32, 2077-2087 (2011).
- Jin, T., Xu, X., Hereld, D. Chemotaxis, chemokine receptors and human disease. Cytokine. 44, 1-8 (2008).
- Xu, X., Jin, T. Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum. J. Vis. Exp. (55), e3128 (2011).
- Chung, B. G., et al. A Gradient-generating Microfluidic Device for Cell Biology. J. Vis. Exp. (7), e271 (2007).
- Rhoads, D. S., Guan, J. L. Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules. Exp. Cell Res. 313, 3859-3867 (2007).
- Park, J., et al. Simple haptotactic gradient generation within a triangular microfluidic channel. Lab on a Chip. 10, 2130-2138 (2010).
- Li Jeon, N., et al. Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat. Biotech. 20, 826-830 (2002).
- Joanne Wang, C., et al. A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues. Lab on a Chip. 8, 227-237 (2008).
- Pujic, Z., Mortimer, D., Feldner, J., Goodhill, G. J. Assays for eukaryotic cell chemotaxis. Combinatorial Chemistry & High Throughput Screening. 12, 580-588 (2009).
- Thery, M., Piel, M. Adhesive micropatterns for cells: a microcontact printing protocol. Cold Spring Harbor protocols. 2009, pdb prot5255 (2009).
- Shen, K., Qi, J., Kam, L. C. Microcontact Printing of Proteins for Cell Biology. J. Vis. Exp. (22), e1065 (2008).
- Shin, Y., et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nat. Protocols. 7, 1247-1259 (2012).
- Qin, D., Xia, Y., Whitesides, G. M. Soft lithography for micro- and nanoscale patterning. Nat. Protocols. 5, 491-502 (2010).
- Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration. Biomicrofluidics. 5, 13412 (2011).
- Hillborg, H., et al. Crosslinked polydimethylsiloxane exposed to oxygen plasma studied by neutron reflectometry and other surface specific techniques. Polymer. 41 (00), 6851-6863 (2000).
- Liu, L., Ratner, B. D., Sage, E. H., Jiang, S. Endothelial Cell Migration on Surface-Density Gradients of Fibronectin, VEGF, or Both Proteins. Langmuir. 23, 11168-11173 (2007).
- Friedl, P., Wolf, K. Plasticity of cell migration: a multiscale tuning model. The Journal of Cell Biology. 188, 11-19 (2010).
- Moazzam, F., DeLano, F. A., Zweifach, B. W., Schmid-Schönbein, G. W. The leukocyte response to fluid. Proceedings of the National Academy of Sciences. 94, 5338-5343 (1997).
- Walker, G. M., et al. Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator. Lab on a Chip. 5, 611-618 (2005).
- Perl, A., Reinhoudt, D. N., Huskens, J. Microcontact Printing: Limitations and Achievements. Adv. Mater. 21, 2257-2268 (2009).
- Salaita, K., Wang, Y., Mirkin, C. A. Applications of dip-pen nanolithography. Nat. Nano. 2, 145-155 (2007).
- Wouters, D., Schubert, U. S. Nanolithography and nanochemistry: probe-related patterning techniques and chemical modification for nanometer-sized devices. Angewandte Chemie (International ed. in English). 43, 2480-2495 (2004).
- Xia, N., et al. Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation. FASEB J. 22, 1649-1659 (2008).
- Oakes, P. W., Beckham, Y., Stricker, J., Gardel, M. L. Tension is required but not sufficient for focal adhesion maturation without a stress fiber template. The Journal of Cell Biology. 196, 363-374 (2012).
Citer Cet Article
Ngalim, S. H., Magenau, A., Zhu, Y., Tønnesen, L., Fairjones, Z., Gooding, J. J., Böcking, T., Gaus, K. Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy. J. Vis. Exp. (74), e50310, doi:10.3791/50310 (2013).