Multimode iiia afm system

Recombinant human Osteopontin (OPN) was purchased from Sigma Aldrich (SRP3131-50UG) and dissolved in MILLI-Q (Millipore, Billerica, MA, USA) water to a concentration of 0.2 μg/μL. Aliquots of the prepared solution were stored at −20 °C in micro centrifuge tubes. Before each experiment, 4 μL of the solution was deposited onto a freshly cleaved mica surface and allowed to dry. The sample was subsequently placed on the scanner of a Multimode IIIa AFM system with Picoforce extension (Bruker Nano: Santa Barbara, CA, USA). Samples were then rehydrated in the Na⁺ buffer solution inside the fluid cell.

The AFM force spectroscopy was done using a Multimode IIIa AFM system with PicoForce (Bruker Nano: Santa Barbara, CA, USA). We used Hydra-All-G (Silicon Nitride cantilevers, gold coated on the reflex side) cantilever (B) from Applied NanoStructutres, Inc. (CA, USA) with a nominal spring constant of 0.045 N/m (manufacture’s value). For proper calibration, the spring constant of the cantilever was determined using the thermal tune method.
The pulls were performed in a 5 × 5 grid with a spacing of 1 µm. For each pull, we followed this procedure: the cantilever was pressed onto the surface with 500 pN force (relative trigger mode) for 3 seconds and then retracted at a speed of 190 nm/s. After each grid was completed, an exchange of the buffer solution (200 µl) was made. For each buffer solution, the grid was repeated 4 times before flushing with the next buffer solution. A total of 100 pulls were therefore recorded per buffer solution. The energy dissipation was calculated from each individual pulling curve using the abovementioned MATLAB data analysis program.

The cell adhesion forces were measured on mammalian HeLa cells using a Multimode IIIa AFM system with PicoForce (Bruker Nano: Santa Barbara, CA, USA). For the adhesion measurements we used tip-less silicon nitride cantilevers (NP-O10, Bruker), functionalized overnight with 2 mg/ml concanavalin A. The spring constant of the cantilever was calibrated using Thermal tune method before performing the experiments (0.068 N/m). The deflection sensitivity of the cantilever (43 nm/V) was also measured before starting the experiments by acquiring force curves on the cell free areas of the glass bottom petri dishes. For measuring the cell adhesion force, the cell was approached towards the surface coated overnight with 10 µg/mL fibronectin with a set force of 1 nN and a speed rate of 10 μm/s. After a contact time of 5 s between the cell and the fibronectin, the sample was retracted 18 μm with a speed rate of 5um/s. The deflection signal of the probe was recorded as a function of retraction until the cell was removed from the surface.