Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young’s (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day.

人们对探索机械介导的生物现象的兴趣日益浓厚，导致具有可变刚度的细胞培养基质和 3D 矩阵成为生物实验室的标准工具。然而，用于确定杨氏（弹性）模量E的方法的可变性以及大多数生物实验室无法获得相关机械工程协议，阻碍了将刚度与生物学结果相关联并比较研究组之间的结果。在这里，我们描述了使用原子力显微镜 (AFM) 力谱测量软 2D 表面和 3D 水凝胶的E 的协议。我们提供了制备带有和不带有封装活细胞的水凝胶的说明，并提供了在 AFM 中安装样品的方法。我们还提供有关如何校准仪器的详细信息，并提供在手动和自动模式（刚度映射）下收集力-位移曲线的分步说明。然后，我们提供有关如何应用 Hertz 或 Oliver-Pharr 模型来计算E 的详细信息，并提供其他说明以帮助用户绘制数据分布和进行统计分析。我们还提供了推断含有封装单细胞或类器官的水凝胶中微分基质重塑活性的说明。我们的方案适用于探测一系列合成和天然衍生的聚合物水凝胶，例如聚乙二醇、聚丙烯酰胺、透明质酸、胶原蛋白或基质胶。尽管样品制备时间会有所不同，但接受过 AFM 入门培训的用户将能够使用该协议在一天内表征两到六个软表面或 3D 水凝胶的机械性能。