用于三维荧光成像的大鼠尾胶原荧光标记
Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging.
作者信息
Doyle Andrew D
机构信息
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda Maryland, USA.
出版信息
Bio Protoc. 2018 Jul 5;8(13). doi: 10.21769/BioProtoc.2919.
Abstract
Rat tail collagen solutions have been used as polymerizable three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen. Here we detail how to generate 3D collagen gels labeled with a fluorescent dye. Furthermore, we go through the steps required to reproducibly generate bright collagen hydrogels that are suitable for live cell 3D imaging techniques.
摘要
大鼠尾胶原溶液已被用作可聚合的三维（3D）细胞外基质（ECM）凝胶，用于单细胞和集体细胞迁移分析以及球体形成。这些3D水凝胶是一种相对便宜、易于使用的模型系统，能够模拟体内许多组织（即皮肤）的物理特性。虽然共聚焦成像技术（如荧光反射和双光子显微镜）能够在3D成像过程中在无荧光的情况下可视化胶原纤维，但其他成像方式需要将荧光染料直接与胶原结合。在这里，我们详细介绍如何生成用荧光染料标记的3D胶原凝胶。此外，我们还介绍了可重复生成适合活细胞3D成像技术的明亮胶原水凝胶所需的步骤。
相似文献
1
Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging.用于三维荧光成像的大鼠尾胶原荧光标记
Bio Protoc. 2018 Jul 5;8(13). doi: 10.21769/BioProtoc.2919.
2
Generation of 3D Collagen Gels with Controlled Diverse Architectures.生成具有可控多样结构的3D胶原蛋白凝胶。
Curr Protoc Cell Biol. 2016 Sep 1;72:10.20.1-10.20.16. doi: 10.1002/cpcb.9.
3
Imaging cells in three-dimensional collagen matrix.对三维胶原蛋白基质中的细胞进行成像。
Curr Protoc Cell Biol. 2010 Sep;Chapter 10:Unit 10.18.1-20. doi: 10.1002/0471143030.cb1018s48.
4
Image-based Characterization of 3D Collagen Networks and the Effect of Embedded Cells.基于图像的 3D 胶原网络特征及其对嵌入细胞的影响。
Microsc Microanal. 2019 Aug;25(4):971-981. doi: 10.1017/S1431927619014570. Epub 2019 Jun 18.
5
Anisotropic dense collagen hydrogels with two ranges of porosity to mimic the skeletal muscle extracellular matrix.具有两种不同孔径范围的各向异性致密胶原水凝胶，以模拟骨骼肌细胞外基质。
Biomater Adv. 2023 Jan;144:213219. doi: 10.1016/j.bioadv.2022.213219. Epub 2022 Nov 24.
6
Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model.时间分辨荧光成像及三维球体模型中癌细胞侵袭的分析。
J Vis Exp. 2021 Jan 30(167). doi: 10.3791/61902.
7
Nanoscale Imaging of Collagen Gels with Focused Ion Beam Milling and Scanning Electron Microscopy.聚焦离子束铣削和扫描电子显微镜对胶原凝胶的纳米级成像
Biophys J. 2016 Oct 18;111(8):1797-1804. doi: 10.1016/j.bpj.2016.08.039.
8
From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels.从宏观到微观：检测左心室脱细胞细胞外基质水凝胶中血管网络形成的成像技术比较
Gels. 2022 Nov 10;8(11):729. doi: 10.3390/gels8110729.
9
Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro.体外胶原纤维形成和细胞外基质组装的延时共聚焦反射显微镜观察。
Biopolymers. 2000 Sep;54(3):222-34. doi: 10.1002/1097-0282(200009)54:3<222::AID-BIP80>3.0.CO;2-K.
10
Polyethylene Glycol-Based Hydrogel as a 3D Extracellular Matrix Mimic for Cytotoxic T Lymphocytes.基于聚乙二醇的水凝胶作为细胞毒性T淋巴细胞的3D细胞外基质模拟物
J Biomed Mater Res A. 2025 Jan;113(1):e37811. doi: 10.1002/jbm.a.37811. Epub 2024 Oct 21.
引用本文的文献
1
Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis.内吞循环对于昼夜节律性胶原纤维生成至关重要，且在纤维化过程中受到破坏。
Elife. 2025 Jan 15;13:RP95842. doi: 10.7554/eLife.95842.
2
Early Stages of Ex Vivo Collagen Glycation Disrupt the Cellular Interaction and Its Remodeling by Mesenchymal Stem Cells-Morphological and Biochemical Evidence.细胞外胶原蛋白糖基化的早期阶段会破坏间充质干细胞的细胞相互作用及其重塑——形态和生化证据。
Int J Mol Sci. 2024 May 26;25(11):5795. doi: 10.3390/ijms25115795.
3
Characterisation and correction of polarisation effects in fluorescently labelled fibres.荧光标记纤维中偏振效应的表征与校正
J Microsc. 2025 May;298(2):185-203. doi: 10.1111/jmi.13308. Epub 2024 Apr 29.
4
A facile fluid pressure system reveals differential cellular response to interstitial pressure gradients and flow.一种简便的流体压力系统揭示了细胞对间质压力梯度和流动的不同反应。
Biomicrofluidics. 2023 Sep 25;17(5):054103. doi: 10.1063/5.0165119. eCollection 2023 Sep.
5
Curved adhesions mediate cell attachment to soft matrix fibres in three dimensions.弯曲的黏附物介导细胞在三维空间中与软基质纤维的附着。
Nat Cell Biol. 2023 Oct;25(10):1453-1464. doi: 10.1038/s41556-023-01238-1. Epub 2023 Sep 28.
6
A Manganese Porphyrin Platform for the Design and Synthesis of Molecular and Targeted MRI Contrast Agents.锰卟啉平台用于设计和合成分子及靶向 MRI 对比剂。
Int J Mol Sci. 2023 May 31;24(11):9532. doi: 10.3390/ijms24119532.
7
3D spheroid culture to examine adaptive therapy response in invading tumor cells.采用3D球体培养法检测侵袭性肿瘤细胞对适应性疗法的反应。
In Vitro Model. 2022;1(6):463-471. doi: 10.1007/s44164-022-00040-x. Epub 2023 Mar 15.
8
Curved adhesions mediate cell attachment to soft matrix fibres in 3D.弯曲粘连介导细胞在三维空间中与软质基质纤维的附着。
bioRxiv. 2023 Mar 19:2023.03.16.532975. doi: 10.1101/2023.03.16.532975.
9
Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.氧化环境中骨髓间充质干细胞对胶原蛋白重塑改变的形态学和定量证据——表没食子儿茶素-3-没食子酸酯的特殊作用
Polymers (Basel). 2022 Sep 22;14(19):3957. doi: 10.3390/polym14193957.
10
Physico-chemical characterization studies of collagen labelled with Ru(II) polypyridyl complex.钌（II）多吡啶配合物标记的胶原蛋白的物理化学表征研究
Heliyon. 2022 Aug 11;8(8):e10173. doi: 10.1016/j.heliyon.2022.e10173. eCollection 2022 Aug.
本文引用的文献
1
Generation of 3D Collagen Gels with Controlled Diverse Architectures.生成具有可控多样结构的3D胶原蛋白凝胶。
Curr Protoc Cell Biol. 2016 Sep 1;72:10.20.1-10.20.16. doi: 10.1002/cpcb.9.
2
Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions.局部3D基质微环境通过收缩力依赖性黏附的时空动态调节细胞迁移。
Nat Commun. 2015 Nov 9;6:8720. doi: 10.1038/ncomms9720.
3
EXTRACTION OF COLLAGEN FROM CONNECTIVE TISSUE BY NEUTRAL SALT SOLUTIONS.用中性盐溶液从结缔组织中提取胶原蛋白。
Proc Natl Acad Sci U S A. 1955 Jan 15;41(1):1-7. doi: 10.1073/pnas.41.1.1.
4
Shape and Movement of Mesenchyme Cells as Functions of the Physical Structure of the Medium: Contributions to a Quantitative Morphology.间充质细胞的形状和运动作为介质物理结构的函数：对定量形态学的贡献
Proc Natl Acad Sci U S A. 1952 Mar;38(3):264-80. doi: 10.1073/pnas.38.3.264.
5
Extraction of collagen from tissues.从组织中提取胶原蛋白。
Nature. 1955 Jul 23;176(4473):163. doi: 10.1038/176163a0.
6
Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro.体外间充质和上皮性癌外植体中协调细胞簇的迁移。
Cancer Res. 1995 Oct 15;55(20):4557-60.
7
Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution.从大鼠尾腱和皮肤制备完整的单体胶原蛋白以及溶液中非螺旋末端的结构。
J Biol Chem. 1976 Oct 10;251(19):6062-7.
Zotero 插件