Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy
Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy
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DOI：
10.1038/nprot.2010.89
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Muller, Daniel J.
中科院分区：
文献类型：
--
作者：
Friedrichs, Jens;Helenius, Jonne;Muller, Daniel J.
基于原子力显微镜（AFM）的单细胞力光谱（SCFS）能够定量研究生理条件下的细胞粘附。SCFS探测单个活细胞与细胞外基质（ECM）蛋白和其他细胞等底物的粘附相互作用。在这里，我们提出了一种使用SCFS研究整合素介导的HeLa细胞与I型胶原的粘附的方案。我们描述了以下步骤：(i)用集素魔豆蛋白A和胶原支架实现AFM悬臂的功能化；（ii）细胞处理和附着在AFM悬臂上；（iii）粘附力的测量；（iv）数据分析和解释。虽然设计用于测量HeLa细胞与胶原的粘附，但该方案可以修改用于其他细胞系和ECM蛋白。与其他SCFS分析（例如，光镊子，生物膜力探针）相比，基于afm的SCFS具有更通用的力检测范围，因此可用于解决更广泛的生物学问题。该方案可在2-3天内完成。
Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here we present a protocol to study integrin-mediated adhesion of HeLa cells to collagen type I using SCFS. We describe procedures for (i) functionalization of AFM cantilevers with the lectin concanavalin A and supports with collagen, (ii) cell handling and attachment to the AFM cantilever, (iii) measurement of adhesion forces and (iv) data analysis and interpretation. Although designed to measure HeLa cell adhesion to collagen, the protocol can be modified for other cell lines and ECM proteins. Compared with other SCFS assays (for example, optical tweezer, biomembrane force probe), AFM-based SCFS has a more versatile force detection range, and it can therefore be used to address a broader range of biological questions. The protocol can be completed in 2-3 d.