Data for "Single molecule force spectroscopy of Hyaluronic Acid"

Pre-processed data obtained by AFM single molecule force spectroscopy on MC3T3 pre-osteoblast cells targeting hyaluronic acid. The data pre-processing consisted in contact point fitting, tip-sample separation correction and drift correction of raw data, all performed in MATLAB (v2016a).The experimental methodology and data processing are described in the author's thesis (Chapter 6). The thesis has been deposited in White Rose eTheses Online (see link in References).Methods* SamplesHyaluronic Acid Binding Protein (HABP) was used to specifically bind Hyaluronic Acid (HA) on the cell surface. Four different samples were employed for experiments, designed as follows:1. HABP/HA: cantilever functionalised with HABP, untreated cell sample;2. BSA/HA: cantilever functionalised with bovine serum albumin (BSA), untreated cell sample;3. untreated/HA: non functionalised cantilever, untreated cell sample;4. HABP/HAase: cantilever functionalised with HABP, cell sample treated with hyaluronidase (HAase).* Cantilever functionalisationLow spring constant cantilevers with pyramidal tip (Olympus) were used for all the experiments (nominal spring constant 0:02 N=m, tip radius 15 nm). The steps of cantilever functionalisation are listed below and were the same for Sample 1 (HABP/HA, functionalisation molecule: HABP), Sample 2 (BSA/HA, functionalisation molecule: BSA) and Sample 4 (HABP/HAase, functionalisation molecule: HABP).The cantilevers used to test cells in Sample 3 (untreated/HA) were not treated, but washed in ultra-pure water prior to experiments.The following activation steps were performed just before the experiments:• deposition of (-SH) groups: cantilevers were oxidised using an ozone cleaner and submerged in 2% (3-Aminopropyl)triethoxysilane (APTES)/ultra-pure water for 15 minutes to depose (-SH) groups on the probe surface;• attachment of intermediate linker molecules: after washing, the cantilevers were submerged in 6 mM Maleimide-PEG-NHS ester/Tris for 30 minutes. This compound bound to the (-SH) groups and exposed NHS esters for subsequent binding to the carboxyl groups of the functionalisationmolecules;• functionalisation: after washing, the functionalisation molecule was bound to the exposed NHS ester groups by submerging the cantilever in 100 nM HABP/Tris solution (Sample 1 HABP/HA and Sample 4HABP/HAase) or 1% BSA/ultra-pure water (Sample 2 BSA/HA) for 1 hour;• blocking: the excess maleimide was quenched with 50 mM2-mercaptoethanol/ ultra-pure water by submerging the cantilevers for 1 minute;• washing: after a final washing, the functionalised cantilevers were kept submerged in ultra-pure water until mounting on the AFM holder.* AFM set-upA NanoWizard 3 Atomic Force Microscope (JPK Instruments AG) coupled to a IX series optical inverted microscope (Olympus) enclosed in a metal box to reduce environmental noise was used for all the experiments.Cells were located through the optical microscope and tested within an area of 10 x 10 μm2. A 16-point grid was drawn and force spectroscopy measurements were obtained on the grid for 3 times to collect a total of 48 data on each cell. The relative set point and the approach velocity were set to 0.5 nN and 2 μm/s respectively.

经原子力显微镜单分子力谱对MC3T3成骨前体细胞进行预处理后获得的数据，旨在针对透明质酸。数据处理过程包括接触点拟合、尖端与样品分离校正以及原始数据的漂移校正，所有操作均在MATLAB（v2016a）软件中完成。实验方法和数据处理方法已在作者论文的第六章中详细描述。论文已存档于White Rose eTheses Online（见参考文献中的链接）。研究方法如下：

* 样本
透明质酸结合蛋白（HABP）被用于特异性结合细胞表面的透明质酸（HA）。实验中使用了四种不同的样本，具体设计如下：
1. HABP/HA：用HABP功能化的悬臂，未经处理的细胞样本；
2. BSA/HA：用牛血清白蛋白（BSA）功能化的悬臂，未经处理的细胞样本；
3. 未处理/HA：未功能化的悬臂，未经处理的细胞样本；
4. HABP/HAase：用HABP功能化的悬臂，经透明质酸酶（HAase）处理的细胞样本。

* 悬臂功能化
所有实验均使用具有金字塔形尖端（Olympus）的低弹簧常数悬臂（标称弹簧常数0.02 N/m，尖端半径15 nm）。悬臂功能化的步骤如下，且对样本1（HABP/HA，功能化分子：HABP）、样本2（BSA/HA，功能化分子：BSA）和样本4（HABP/HAase，功能化分子：HABP）均相同。用于测试样本3（未处理/HA）细胞的悬臂未经处理，但在实验前用超纯水清洗。以下激活步骤在实验前进行：
• (-SH)基团的沉积：使用臭氧清洁剂氧化悬臂，并将其浸泡在2%的（3-氨丙基）三乙氧基硅烷（APTES）/超纯水中15分钟，以在探针表面沉积(-SH)基团；
• 中间连接分子的附着：清洗后，悬臂被浸泡在6 mM Maleimide-PEG-NHS酯/Tris中30分钟。该化合物与(-SH)基团结合，并暴露NHS酯，以便随后与功能化分子的羧基结合；
• 功能化：清洗后，功能化分子通过将悬臂浸泡在100 nM HABP/Tris溶液中（样本1 HABP/HA和样本4 HABP/HAase）或1% BSA/超纯水中（样本2 BSA/HA）1小时，与暴露的NHS酯基团结合；
• 阻断：通过将悬臂浸泡在50 mM 2-巯基乙醇/超纯水中1分钟，用50 mM 2-巯基乙醇/超纯水淬灭过量的Maleimide；
• 清洗：最终清洗后，功能化悬臂被浸泡在超纯水中，直至安装到原子力显微镜的夹具上。

* 原子力显微镜设置
使用配备IX系列光学倒置显微镜（Olympus）的NanoWizard 3原子力显微镜（JPK Instruments AG），该显微镜被封装在一个金属箱中以减少环境噪声，用于所有实验。通过光学显微镜定位细胞，并在10 x 10 μm²的区域进行测试。绘制了一个16点的网格，并对网格进行三次力谱测量，以收集每个细胞的总共48个数据点。相对设定点和接近速度分别设置为0.5 nN和2 μm/s。