Cell surface stiffness was examined by indentation assay using an AFM cantilever as described previously55 (link), 56 (link). Briefly, force probe was prepared by attaching a glass bead (ca. 100 μm diameter) to a tipless silicon cantilever (450 μm long, 50 μm wide, 2 μm thick; nominal spring constant 0.02 N/m; TL-CONT, Nanosensors) using two component Araldite epoxy glue. The cells were set on a plastic culture dish, and pressed from the apical side with a force of 20 nN (the approach and retraction speeds were set to 1.0 μm/s) by the force probe. The measurement using a contact mode was carried out with the Nano-Wizard system with the Cell-Hesion module (JPK), and the data analysis was done with the JPK data processing software (JPK Instruments), where the cell elasticity (Young’s modulus) in the apical region was estimated from a force–distance curve using the Hertz model. The cells were cultured in Leibovitz’s L-15 medium (Thermo Fisher). Nocodazole treatments were given for 60 min at 37 °C before the measurements. All measurements were performed at room temperature.
Tl cont
Manufactured by Nanosensors
Sourced in Switzerland
The TL-CONT is a laboratory equipment designed for temperature control applications. It provides precise and reliable temperature regulation for various experimental setups. The core function of the TL-CONT is to maintain a specified temperature within a defined range, ensuring consistent and reproducible experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Cellhesion module, by Bruker (1 mentions)
Jpk data processing software, by Bruker (1 mentions)
Nano wizard system, by Bruker (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Silica microspheres, by Thermo Fisher Scientific (1 mentions)
Te2000 u, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Glass beads, by Polysciences (1 mentions)
Jpk bioafm nanowizard 3, by Bruker (1 mentions)
4 protocols using tl cont
1
Cell Surface Stiffness Measurement Using AFM
2
Nanomechanical Characterization of SKOV-3 Cells
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The SKOV-3 cells were seeded in Petri dishes, coated with hydrogel substrates of different stiffness (Matrigen, United States) for 3 days. To probe the nanomechanical properties of the cells, we employed an AFM instrument (5,500; Keysight, Santa Rosa, CA, United States) combined with an inverted microscope (TE2000U; Nikon, Tokyo, Japan). As the 0.5 kPa hydrogel was too soft for AFM experiments, we instead used hydrogels with stiffness values of 4, 25, and 50 kPa for this test. Each cell was probed by recording the approach part of the force-distance curve at the central region of the cytoplasm with a frequency of 1 Hz. According to our previous method (Shi et al., 2010 (link)), silica microspheres (Thermo Fisher Scientific, United States) with a diameter of approximately 10 μm were attached to the tipless cantilever with a typical spring constant (k) of about 0.2 Nm–1 (TL-CONT, NANOSENSORS, Neuchatel, Switzerland). The force-distance curves were converted into force-indentation curves and fitted to the spherical Hertz model to calculate Young’s modulus of the cells. Before AFM indention testing, the spring constant of the cantilever was determined using the thermal tune method. According to the slope of the force-distance curves acquired on the glass substrate, the deflection sensitivity of the cantilever was measured in the fluid.
3
Quantifying Cell Mechanics Using AFM Force Curves
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An AFM (Agilent 5500) with an inverted microscope (Nikon Eclipse Ti) was used to observe and locate the relative positions of the probe and cell, and obtain the force curves. In our experiments, an AFM probe consisting of a silicon nitride tipless cantilever (TL-CONT, Nanosensors) and coupled to 10-μm-diameter SiO2 particles with experimentally determined spring constants was used to capture the force curve. The spring constant of the AFM probe was experimentally determined to be 0.095 N/m.
As is shown inFigure 1A , AFM tips were made to approach the cell surface at a rate of 20 μm/s, produce an indentation with a depth of about 1 μm, and then to remain stationary for about 3 s until the force relaxation signal gradually stabilized. Distance, time and deflection data for the whole process were recorded and used in subsequent theoretical calculations and data analysis.
Thirty cells for each type of cell morphology were selected. For each cell, three force curves were collected and averaged. The experiment of each type of cell was completed within 1 h.
As is shown in
Thirty cells for each type of cell morphology were selected. For each cell, three force curves were collected and averaged. The experiment of each type of cell was completed within 1 h.
4
Biomechanical Assessment of Mouse Embryonic Cartilage and Muscle
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E11.5 mouse embryos were extracted and snap-frozen in O.C.T. compound with liquid nitrogen. Those embryos were then serially cryo-sectioned by a microtome with a thickness of 20 μm. To identify nascent cartilage and muscle regions for AFM measurement, we performed immunofluorescence staining (cartilage: Sox9, muscle: MyoD) and in situ mRNA hybridization (cartilage: Col2a1, muscle: MyoD) for one-before and one-after cryo-sections, respectively. Finally, those staining results for those neighboring cryo-sections were collated with AFM results. For AFM-based tissue indentation experiment, the cryo-section was thawed and the nuclei were immediately stained with Hoechst 33342 in PBS(-). In this study, AFM system (JPK BioAFM NanoWizard 3; Bruker Nano GmbH., IX81; Olympus Co.) was used and AFM cantilevers (TL-CONT; spring constant 0.2 N/m; NANOSENSORS) were modified with glass beads (15 μm-diameter; Polysciences. Inc.). Spring constant of the AFM cantilever was calibrated with a thermal noise method. Piezo displacement speed was set to be 3 μm/s. As a result of tissue indentation, force (F) versus indentation depth (h) curves were obtained and slope (nN/μm) was evaluated for each curve by linear regression. Sample points within the force range of 750 pN≤F≤ 1000 pN were used for the evaluation. After AFM, the sample was immunostained for Sox9 and MyoD.
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