Jpk nanowizard 4 bioscience afm

A JPK NanoWizard 4 BioScience AFM (JPK Instruments, Berlin, Germany) was used to optically align the probe to the cells. The probes used in this study were HYDRA6V-100NG (AppNano, CA, USA) with a nominal spring constant of 0.292 N/m. During the indentation process, the loading and retraction speeds of all experiments were maintained at ~2.5 μm/s to avoid viscosity effects. Measurements were made in PBS at room temperature, and the cells were plated on the bottom of the cell culture dish. After transfection of the LINC00472 overexpression vector for 24 h, cells were washed twice with PBS, fixed with 2% glutaraldehyde for 45 s and a 4% polymethanol solution for 20 min. Cells were washed five or more times with PBS and maintained in an appropriate amount of PBS for subsequent AFM scanning. The indentation depth was at least 1 mm to better simulate physiologically occurring deformations. Imaging was performed using the QI mode, and images of the AFM scan were analyzed using JPK image processing software. The force and indentation curves from each measurement were analyzed using a Hertz model to obtain the Young’s modulus for each cell.

AFM studies were performed on a JPK NanoWizard 4 BioScience AFM (JPK Instruments, Germany) integrated with an iX81 optical microscope (Olympus, Belgium). SH-SY5Y cells were seeded on a glass bottomed dish and cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were exposed to 200 μl of the nanoparticle solutions (60 μg ml⁻¹ of GNPs, GNPs–LA, GNPs–α-Syn and the mixture GNPs–LA/GNPs–α-Syn) in the cell growth medium for 24 h and 72 h prior to the AFM measurements. High-resolution topographical images were obtained in Quantitative Imaging mode (QI) using a silicon tip with a nominal radius of 20 nm, spring constant of 0.02 N m⁻¹ and resonance frequency of 7–10 kHz. Cell elasticity measurements were performed in the Force Spectroscopy mode of JPK and spherical tips of 15 μm were used for this purpose. The data were processed by JPK software and the Young's modulus values were extracted using the Hertz model for the spherical indenter. The spring constant of the cantilever was measured before each experiment using the thermal noise method in the JPK software. All AFM studies were performed under physiological conditions at 37 °C and in the appropriate cell medium.