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Data for "Single molecule force spectroscopy of Hyaluronic Acid"
dataset
posted on 2018-10-23, 10:38 authored by Stefania Marcotti, Damien LacroixDamien Lacroix, Gwendolen ReillyGwendolen ReillyPre-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).
* AFM set-up
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.
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
* Samples
Hyaluronic 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 functionalisation
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).
Hyaluronic 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 functionalisation
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).
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 functionalisation
molecules;
• 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
HABP/HAase) or 1% BSA/ultra-pure water (Sample 2 BSA/HA) for 1 hour;
• blocking: the excess maleimide was quenched with 50 mM
2-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.
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 functionalisation
molecules;
• 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
HABP/HAase) or 1% BSA/ultra-pure water (Sample 2 BSA/HA) for 1 hour;
• blocking: the excess maleimide was quenched with 50 mM
2-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-up
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.
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.
