For AFM cell stiffness measurements, Vero cells were plated at 60% confluence in 50-mm glass-bottom dishes (GWST-5040, WillCo Wells BV) the day before infection, infected, and fixed as described before. Live cell probes (PFQNM-LC, Bruker AFM probes) were used for all experiments. The probes were precalibrated for spring constant (nominal of 0.08 N/m), and deflection sensitivity was calibrated at the start of each experiment. The force applied to the cells was kept constant throughout the experiments, with typical values ranging between 150 and 300 pN. Force curves were fitted to a Hertz model where Rc is the radius of tip curvature, v is the sample’s Poisson’s ratio, E is the Young’s modulus, and δ is the indentation depth. Curve fitting and Young’s modulus calculation were performed using nanoscope analysis.
Pfqnm lc
Manufactured by Bruker
The PFQNM‐LC is a high-resolution atomic force microscopy (AFM) mode designed for the Bruker Dimension series of AFM instruments. It enables the quantitative measurement of nanomechanical properties, such as adhesion, elasticity, and viscoelasticity, of samples in liquid environments.
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Lab products found in correlation
3 protocols using pfqnm lc
1
AFM Measurements of Vero Cell Stiffness
2
AFM Probe Functionalization for Single-Molecule Studies
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NHS‐PEG27‐acetal linkers were used to functionalize AFM probes.[58 ] AFM tips (PFQNM‐LC, Bruker) were first immersed in chloroform for 10 min dried with a stream of filtered nitrogen, cleaned for 10 min using an ultraviolet radiation and ozone (UV‐O) cleaner (Jetlight) and incubated during 2 h in a desiccator under argon with a tray with APTES and another tray with triethylamine (30 µL APTES and 10 µL triethylamine). After removing the APTES and trimethylamine trays, the tips were left inside the dessicator for 2 days to cure the APTES coating. To ensure a low grafting density of the linker on the AFM tip, acetal‐PEG24‐NHS (3.3 mg) was diluted in chloroform (0.5 mL) and trimethylamine (30 µL). The cantilevers were immersed for 2 h in this solution, washed three times with chloroform, and dried with nitrogen. Cantilevers were then immersed for 1 h in Gly10Lys (1 × 10−3m ) and washed three times with milliQ water. The tips were then immersed for 1 h at 37 °C in a freshly prepared solution of θ‐toxin (10 × 10−6m ) and sortase A (10 × 10−6m ), washed three times with Tris‐buffer (50 × 10−3m Tris, 150 × 10−3m NaCl, 10 × 10−3m CaCl2, pH 7.5) and stored at 4 °C in the same buffer. Cantilevers were used in AFM experiments the same day they were functionalized.
3
Atomic Force Microscopy of Biomolecular Surfaces
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AFM images were recorded in PBS with a BioScope Resolve microscope (Bruker) equipped with a 100 × 100 × 15 μm (x, y, z) piezoelectric scanner. AFM cantilevers (PFQNM‐LC from Bruker) had nominal spring constants of ≈0.08 N/m, had resonance frequencies of ≈60 kHz in buffer, and carried a SiO2 stylus with a nominal apex radius of ≈65 nm. The cantilevers were functionalized with NH2 groups, then coated with heterobifunctionalized 27‐unit long PEG linkers (NHS‐PEG27‐maleimide), and quenched with β‐mercaptoethanol to lower the adhesion to imaged surfaces (Wildling et al, 2012 ). Images were taken using force‐distance curve‐based AFM (Dufrêne et al, 2013 ; PeakForce mode, Bruker) in buffer solution (20 mM Hepes, 50 mM NaCl, pH 7.4) at room temperature. Imaging parameters included a scan rate of 0.4–0.6 Hz, sampling rate of 2 kHz, maximum imaging force of ≈200–300 pN and oscillating the AFM stylus at a vertical amplitude of ≈400 nm. For each topographic pixel (512 × 512 px), the force‐feedback loop limited the imaging force by regulating the stylus sample distance, which was taken to reconstruct the AFM topography. Topographs were flattened using built‐in processing tools. Cross‐sectional profiles were analyzed using ImageJ v1.43 (National Institutes of Health) and plotted using GraphPad Prism 7.
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