Data for "Single molecule force spectroscopy of Hyaluronic Acid"
2018-10-23T10:38:35Z (GMT) by
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).<br><br><div>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).<br></div><div><br></div><div><u><b>Methods</b></u></div><div><u></u><b></b><br></div><div>* <u>Samples</u><br>Hyaluronic Acid Binding Protein (HABP) was used to specifically bind Hyaluronic Acid (HA) on the cell surface. <br>Four different samples were employed for experiments, designed as follows:<br>1. HABP/HA: cantilever functionalised with HABP, untreated cell sample;<br>2. BSA/HA: cantilever functionalised with bovine serum albumin (BSA), untreated cell sample;<br>3. untreated/HA: non functionalised cantilever, untreated cell sample;<br>4. HABP/HAase: cantilever functionalised with HABP, cell sample treated with hyaluronidase (HAase).<br><br>* <u>Cantilever functionalisation</u><br>Low spring constant cantilevers with pyramidal tip (Olympus) were used for all the experiments (nominal spring constant 0:02 N=m, tip radius 15 nm). </div><div>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).<br>The cantilevers used to test cells in Sample 3 (untreated/HA) were not treated, but washed in ultra-pure water prior to experiments.<br>The following activation steps were performed just before the experiments:<br>• 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;<br>• 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 functionalisation<br>molecules;<br>• 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 4<br>HABP/HAase) or 1% BSA/ultra-pure water (Sample 2 BSA/HA) for 1 hour;<br>• blocking: the excess maleimide was quenched with 50 mM<br>2-mercaptoethanol/ ultra-pure water by submerging the cantilevers for 1 minute;<br>• washing: after a final washing, the functionalised cantilevers were kept submerged in ultra-pure water until mounting on the AFM holder.</div><div><br>* <u>AFM set-up</u><br>A 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.<br></div><div>Cells were located through the optical microscope and tested within an area of 10 x 10 μm<sup>2</sup>. 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.<br><br></div>