The stiffness of cells was measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica), as described previously50 (link),51 (link). The point-and-shoot procedure (NanoScope software; Bruker) was used to measure cell stiffness. All cells were kept in CO2-independent cell culture medium during the measurement. A fluorescent 10 μm polystyrene bead (Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N m−1 (NP-S type D; Bruker). Indentations were performed using the single force option with a total indentation depth of 50–100 nm. To obtain cell stiffness values from force curves, PUNIAS software was used as described previously50 (link),51 (link). Multiple force displacement curves (at five different locations) were fitted to the Hertz model to calculate cell cortical stiffness (Young’s modulus).
Np s type d
Manufactured by Bruker
The NP-S type D is a laboratory instrument designed for the characterization of nanoparticles. It utilizes the principle of dynamic light scattering to measure the size distribution of particles suspended in liquid samples.
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Lab products found in correlation
4 protocols using np s type d
1
Cell Stiffness Measurement using AFM
2
Nanoindentation for Substrate Stiffness Measurement
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Substrate stiffness was measured by nanoindentation under an atomic force microscope (Bruker Nanoscope) using the “point and shoot” procedure (Nanoscope software, Bruker). A fluorescent polystyrene bead (φ = 10 μm, Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N/m (NP-S type D, Bruker). The system was calibrated in cell-free medium at 37 °C prior to each experiment by measuring the deflection sensitivity when pressing the cantilever onto a glass coverslip, which allowed the cantilever spring constant to be determined using the thermal noise method38 (link). For each gel, indentation force curves at 30 different locations on the gels were acquired. Before and during indentation experiments gels were kept in medium in 37 °C. To address local stiffness changes generated by cells on protein gels and PAAm gel, we applied spatially resolved AFM nanoindentation in live-cell culture by probing the matrix around single cells. To obtain stiffness values from force curves we used the PUNIAS software (http://punias.free.fr ). Specifically, we corrected for baseline tilt, and used the linear fitting option for the Hertz model with a Poisson ratio of 0.5 on the indentation curve.
3
Probing Collagen I Fibrillar Matrices with AFM
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The fibrillar matrices of Coll I were reconstituted and probed in 1× PBS by an atomic force microscope (AFM, BioScope Catalyst, Bruker Corporation) coupled to a fluorescence microscope (TCS SP5 II, Leica). Prior to experiments, a polystyrene microsphere (15 µm diameter; Polysciences, Inc.) was glued to a cantilever (NP‐S type D nominal spring constant of 0.08 N m−1, Bruker Corporation) by a two‐component polyurethane glue (Bison International). The spring constant of the bead‐functionalized cantilever was calibrated per manufacturer's instructions. Matrix rigidities were quantified by repeatedly bringing the cantilever into contact with the surface of the specimens at an identical position (contact force ≤2 nN; approach‐retraction distance of 14 µm; approach velocity of 10 µm s−1). In each sample group, at least 16 force‐distance curves at three different positions were acquired from three independent experiments (Figure S12 , Supporting Information).
4
Tuning Hydrogel Stiffness via MeHA Concentration
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In order to show the possibility of changing stiffness for MeHA hydrogel, different MeHA concentration was used to obtain gels with a range of crosslinking densities. The stiffness of gels was measured by nanoindentation under an atomic force microscope (Bruker Nanoscope) using the “point and shoot” procedure (Nanoscope software, Bruker) as we reported previously48 (link). A fluorescent polystyrene bead with a 10-μm diameter (Invitrogen) was glued to silicon nitride cantilevers with a nominal spring constant of 0.06 N m−1 (NP-S type D, Bruker). We calibrated the system in a cell-free medium at 37 °C prior to each experiment by measuring the deflection sensitivity when pressing the cantilever onto a glass coverslip, which allowed the cantilever spring constant to be determined using the thermal noise method. For each MeHA gel, indentation force curves at six different locations on the gels were acquired. Before and during indentation experiments, gels were kept in PBS at 37 °C. To obtain the stiffness values from force curves, we used the PUNIAS software (http://punias.free.fr ). Specifically, we corrected for baseline tilt, and used the linear fitting option for the Hertz model with a Poisson ratio of 0.5 on the indentation curve.
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