Cells were plated at the density of 3 × 103 cells/cm2, and cellular stiffness on top of the nucleus and the cytosol was measured using atomic force microscopy (AFM) indentation method 29 (link)–31 (link) by the JPK NanoWizard II AFM system (JPK Instruments, Berlin, Germany). The cantilever was the tipless cantilever (ARROW-TL1-50; NanoWorld, Neuchatel, Switezerland), which conjugated with a polystyrene bead (with diameter of 5 μm). Before each measurement, cantilever was calibrated and possessed a mean spring constants ranging from 0.01 to 0.03 nN/nm. For stiffness measurements, 1 nN indentation was applied to each cell. The indentation depth was about 0.5 μm to avoid the substratum effect. The approaching speed of the cantilever to cell surface was set at 1 μm/sec. Force-distance curves were collected by landing the bead on region of interests and processed using JPK IP software. To extract the mechanical properties of cells from force-distance curves, the data were analysed by the Hertz model.
Arrow tl1 50
Manufactured by NanoWorld
Sourced in Switzerland
The ARROW-TL1-50 is a high-precision laser system designed for research and industrial applications. It emits a narrow, collimated beam of light with a wavelength of 1050 nanometers. The system offers a maximum output power of 50 watts and is capable of operating in continuous wave or pulsed modes.
Automatically generated - may contain errors
Lab products found in correlation
Csc12, by MikroMasch (1 mentions)
Jpk nanowizard 2 afm with biocell, by Bruker (1 mentions)
Axio observer, by Zeiss (1 mentions)
Cell tak, by Corning (1 mentions)
Cytodex 3, by GE Healthcare (1 mentions)
Cytopore1, by GE Healthcare (1 mentions)
Cellhesion 200, by Bruker (1 mentions)
Triton x 100, by Merck Group (1 mentions)
6 protocols using arrow tl1 50
1
Atomic Force Microscopy for Cellular Stiffness
2
Measuring Tissue and Gel Stiffness via AFM
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For measurements of the stiffness of cells or tissues, the NanoWizard® II AFM with BioCell (JPK Instrument) was equipped and manipulated as previously described 26 (link). Tumor tissues were embedded in OCT compound and then sliced into sections with 30 µm in thickness. The sections were subsequently put onto glass slides and immersed in PBS supplemented with protease inhibitor. The 20 μm (in diameter) polystyrene bead-modified tip-less cantilever (CSC12, MikroMasch) with nominal spring constant 0.3N/m was used to generate 3nN applied force with 1 µm/second approaching/retracting speed (to avoid the effects of hydrodynamics on AFM assessment) on pancreatic tissues. After cells were cultured in collagen gel on 6-well inserts, the stiffness of the collagen gel was measured by AFM. The 5 μm-diameter polystyrene bead-modified tip-less cantilever (ARROW-TL1-50, NanoWorld) with nominal spring constant 0.03N/m was used to apply 1nN with 1 µm/second approaching/retracting speed on collagen gels. All the force-displacement curves obtained from pancreatic tissues and collagen gels were analyzed using the JPK package software, from which the effective Young's modulus of the pancreatic tissues and collagen gels were calculated based on the Hertz model 27 (link). At least 60 points of indentation results were collected in each experiment.
3
Measuring Kidney Tissue Mechanics via AFM
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For measurements of mechanical properties of tissue/cell, JPK NanoWizard II AFM with BioCell (JPK Instruments, Berlin, Germany) was equipped and manipulated as previously described. 43 Fresh kidney tissue samples were sliced at a thickness of 100 mm with a microtome. Tissue slices were glued to a glass coverslip with a small drop of nail polish, and only the intact side of the cortex was immediately subjected to AFM measurements. Tipless cantilevers (Arrow-TL1-50; Nanoworld, Neuchâtel, Switzerland) modified with 5-mm diameter polystyrene bead were used to measure tissue and cells. The spring constants of all cantilevers were calibrated via the thermal noise method in liquid before each measurement and valued 0.03 N/m. The indenting force was set at 1 nN. Force-distance curves were collected and calculated with JPK package software version 4.6.62 (JPK Instruments), which was based on the Hertz model.
4
Measuring Gel Substrate Elasticity
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Substrate elasticity of uniform gels was measured by using a JPK NanoWizard II AFM system installed above the stage of an inverted light microscope (Zeiss Axio Observer) in a custom-built anti-noise, anti-vibration system. A 5 µm (in diameter) polystyrene bead-modified tip-less cantilever (ARROW-TL1-50, NanoWorld, US) was utilized. The spring constants, calibrated by a thermal noise-based method, were at a range of 0.02 to 0.08 N/m for all cantilevers. All indentation depth curves were calculated using the manufacturer’s software (Hertz model, JPK instruments, Berlin, Germany). The average value of Young’s modulus of tensile elasticity was acquired from 25 measurements for each independent experiment.
5
Microcarrier Elasticity Comparison to Bone Marrow
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The Young’s Modulus of Cytopore1 (GE Healthcare, 17-0911-01), Cytodex3 (GE Healthcare, 17-0485-01), and two types of Microcarriers (CytoNiche, Beijing, Microcarriers-W01, Microniche)25 (link)–28 (link) were measured by AFM and compared with the elasticity of natural bone marrow (Using Yong Modulus in the range of 0.3–24.7 Kpa)42 (link)–44 (link). The microcarriers were dispersed with Cell-Tak (Corning) and attached to a coverslip, while samples attached to the coverslip were mounted on an inverted fluorescence microscope (Zeiss Observer A1 stand) and measured using an AFM module CellHesion® 200 (JPK Instruments). The silicon tipless cantilever (ARROW-TL1-50, NANOWORLD) that was used to indent the samples had a nominal spring constant of 0.03 N/m and was attached with a silicon microsphere (20 µm in diameter) on the cutting edge. For each sample, AFM indentations at different locations (n ≥ 30) of the cell were performed with ~0.6 nN force at a 10 µm/s displacement rate. The effective Young’s modulus was obtained by fitting the force-displacement curve to the Hertz/Sneddon model.
6
Indentation Assay with Silica Bead-Tipped AFM
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Indentations on cells were performed using an AFM equipped with a cantilever of nominal resonance frequency f0 = 6 kHz and a spring constant k = 0.03 N/m (Arrow-TL1-50, NanoWorld AG, Neuchatel, Switzerland). A spherical silica bead (radius: 3.6 µm, Kisker Biotech, PSI-5.0, surface plain, Steinfurt, Germany) was glued (UHU plus Endfest 300; UHU, Bühl, Baden, Germany) onto the cantilever tip as previously described [26 (link)]. AFM indentations were performed at room temperature with a cantilever speed of 0.5 µm/s for approach and 2 µm/s for retract. Indentations were repeated six times for each position with 2 s pauses between measurements. A sampling rate of 2 kHz and a force set point of 1.5 nN were used. Before each measurement, cantilever spring constants and sensitivities were calibrated in culture medium using the thermal noise method [34 (link)], and the slope of a sample force–distance curve was recorded on a stiff substrate as usual [34 (link)] (glass Petri dish in this case). Cantilevers were equilibrated for 30 min in culture medium before calibrations. AFM control measurements in chambers without cells were performed in 2% detergent solution in water (Triton X-100, Sigma-Aldrich Chemie GmbH, München, Germany). No additional CO2 supply was used.
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