Imaging cells in three-dimensional collagen matrix.
Imaging cells in three-dimensional collagen matrix.
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DOI：
10.1002/0471143030.cb1018s48
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发表时间：
2010-09
影响因子：
--
通讯作者：
Matsumoto, Kazue
中科院分区：
文献类型：
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作者：
Artym, Vira V;Matsumoto, Kazue
使用体外三维（3D）胶原基质来模拟体内细胞环境已经变得越来越流行，并且正在拓宽我们对细胞过程和细胞- ECM相互作用的理解。为了在体外3D胶原基质中研究细胞，必须使细胞蛋白和胶原基质都可视化。在本单元中，作者描述了该协议，并提供了3D胶原凝胶中细胞免疫标记的故障排除，以使用高分辨率荧光共聚焦显微镜定位和可视化细胞蛋白。然后，作者描述了共聚焦反射显微镜作为一种直接成像的三维纤维胶原基质的技术，讨论了该技术的优点和缺点。他们还提供了所需的仪器设置，同时成像的细胞蛋白与荧光共聚焦成像和3D胶原纤维与共聚焦反射显微镜。此外，作者提供了一种“细胞培养”技术的方案，以在高分辨率共聚焦成像所需的3D胶原基质中制备细胞培养物。
The use of in vitro three-dimensional (3D) collagen matrices to mimic an in vivo cellular environment has become increasingly popular and is broadening our understanding of cellular processes and cell - ECM interactions. To study cells in in vitro 3D collagen matrices, both cellular proteins and the collagen matrix must be visualized. In this unit, the authors describe the protocol and provide troubleshooting for immuno-labeling of cells in 3D collagen gels to localize and visualize cellular proteins with high-resolution fluorescence confocal microscopy. The authors then describe confocal reflection microscopy as a technique for direct imaging of 3D fibrilar collagen matrices by discussing the advantages and disadvantages of the technique. They also provide instrument settings required for simultaneous imaging of cellular proteins with fluorescence confocal imaging and 3D collagen fibrils with confocal reflection microscopy. Additionally, the authors provide protocols for a “cell sandwiching” technique to prepare cell cultures in 3D collagen matrices required for high resolution confocal imaging.