Nanowizard 4 bioscience afm

Atomic force microscopy (AFM) was employed to record the topography of treated bacterial cells for the qualitative assessment of AuNPs-mediated bacterial treatment. Representative strains of E. coli and P. aeruginosa were resuspended in distilled water (OD₆₀₀ ~ 0.1) and incubated with indicated concentrations of AuR NPs at 37 °C for 1 h. Then, 200 μL bacterial samples were transferred to the mica surface previously functionalized with 0.5% APTES. The attachment of bacterial cells to the mica surface was achieved during 20 min of incubation. Images of bacterial cell surface were collected using a Nano Wizard 4 BioScience AFM (JPK Instruments, Berlin, Germany) operated in Quantitative Imaging Mode. MSNL cantilevers (Bruker, MA, USA) with a nominal spring constant equal to 0.1 N/m were employed. The bacterial cells were located using an optical microscope to collect the topography and adhesion images. Then, 10 μm × 10 μm scanning was performed with the resolution of 128 pixels per line.

The morphological changes of two of the tested strains (A6 and A9) were analyzed by AFM using a NanoWizard 4 BioScience AFM (JPK instruments AG, Germany). The protocol was used as previously described^{20 (link)}. Briefly, bacterial strains were treated as previously described, in the presence of the peptide at MIC and MIC/2, for 18–24 h, with continuous agitation. Then, the cells were centrifuged, washed in sterile phosphate buffer saline and re-suspended in deionized sterile water. Finally, cell suspensions (10 μL of each condition) were applied, on circular mica disks (V1 Ruby Muscovite) and left to dry prior to AFM imaging. An area of 4 μm × 4 μm was scanned and the images were analyzed with Gwyddion software to obtain the morphological characteristics (diameter, height and roughness-Ra).

The stiffness of cells after 24 h stimulation with 1 µg/mL LPS was measured by indentation using atomic force microscopy (Nanowizard 4 Bioscience AFM, JPK Instruments, Germany). Adenocarcinomic human alveolar basal epithelial cells (A549 ATCC^® CCL-185™) were cultured in high-glucose DMEM supplemented with 10% FBS at 37 °C with 5% CO₂, placed on Ø 35 mm Petri dishes and allowed to attach and spread for 24 h before simultaneous stimulation with 1 µg/mL LPS (E. coli 026:B6) and treatment with 5 µg/mL of PBP10 or PBP10-based nanosystems. Immediately before the experiment, cells were placed in a CO₂-independent buffer (Thermo Fisher Scientific, USA) to prevent changes in pH of the cellular environment during analysis. Elasticity measurements were taken with AFM working in force spectroscopy mode in liquid conditions, and cantilevers (ORC8, Bruker) with a spring constant of 0.1 N/m were used. From each tested cell, up to 64 force-indentation curves were collected in a grid of 8 × 8 pixels corresponding to a scan area of 10 × 10 µm with a maximal force of 2 nN. For indentation measurements, more than 1100 force-distance curves were recorded for each group from at least ten different cells. To determine the apparent Young’s modulus of different cells, force-indentation curves were fit to the Hertz contact model.