Particle concentration and size distribution of hiPSC-EVs were measured by NanoSight NS300 analyzer (Malvern Pananalytical, Malvern, UK) based on the nanoparticle tracking analysis (NTA). Atomic force microscopy (AFM) analysis of hiPSC-EV samples was performed by BioScope Catalyst AFM system (Brüker, Billerica, MA, USA), as previously described [13 (link), 15 (link)]. For flow cytometry analysis, hiPSC-EVs were stained with RNASelect dye (Thermo Fisher Scientific) and antibodies against CD9, CD34, CD45, CD63, CD81, CD90, CD105, Tra-1-60, KDR, lymphocyte function-associated 1 protein (LFA-1), and stage-specific embryonic antigen-4 (SSEA-4) and further analysis was performed with an Apogee A50-Micro flow cytometer (Apogee Flow Systems, Hemel Hempstead, UK). In addition, stained hiPSC-EVs were also imaged by ImageStreamX Mk II imaging flow cytometer, using 60× objective magnification and IDEAS Software (Luminex Corp., Austin, TX, USA.). Moreover, western blotting analysis was performed to compare relative levels of expression of CD9, CD63, syntenin, calnexin and β-actin in lysates obtained from hiPSC-EVs and their parental cells.
Bioscope catalyst afm system
Manufactured by Bruker
Sourced in United States
The BioScope Catalyst AFM system is a high-performance atomic force microscope designed for life science and materials science applications. It provides nanoscale topographical and mechanical characterization of samples in various environments, including air, liquid, and controlled temperature and humidity conditions.
Automatically generated - may contain errors
Lab products found in correlation
Plan apochromat, by Zeiss (2 mentions)
Plan apochromat objective lens, by Zeiss (2 mentions)
Petri dish heating stage, by Bruker (2 mentions)
Axiovert 200m microscope system, by Zeiss (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Nanosight ns300 analyzer, by Malvern Panalytical (1 mentions)
Icam 1, by Thermo Fisher Scientific (1 mentions)
Hq csc38 tipless cr au, by MikroMasch (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Nanoscope analysis, by Bruker (1 mentions)
Silicon nitride cantilever, by Bruker (1 mentions)
Eclipse te200, by Nikon (1 mentions)
9 protocols using bioscope catalyst afm system
1
Characterization of hiPSC-Derived Extracellular Vesicles
2
Measuring HUVEC Elasticity on Scaffolds
3
PFT-AFM Nanomechanical Mapping of Stereocilia
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PFT-AFM images were obtained using a Bruker BioScope Catalyst AFM system (Bruker) mounted on an inverted Zeiss Axiovert 200M opti-cal microscope equipped with a 40× objective (0.95 numerical aperture, Plan-Apochromat, Zeiss) and a confocal laser scanning microscope (LSM 510 META, Zeiss). For detailed descriptions of the PFT-AFM nanomechanical mapping, generation and analysis of Young’s modulus maps, and analysis of the pivotal stiffness of stereocilia, see SI Appendix, Supplementary Materials and Methods .
4
Characterizing T Cell Mechanics via AFM
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hCD4+ T cells were plated on a glass-bottom dish (Willco Wells) precoated with either ICAM1 (Life Technologies) or 0.01% poly-l-lysine (Sigma-Aldrich) and immersed in culture media solution (Life Technologies). Force spectroscopy AFM experiments were performed using a Bruker Bioscope Catalyst AFM system (Bruker) mounted on an inverted Axiovert 200 M microscope (Zeiss) equipped with a confocal laser-scanning microscope 510 Meta (Zeiss) and a ×40 objective lens (0.95 NA, Plan-Apochromat, Zeiss). The hybrid microscope instrument was placed on an acoustic isolation table (Kinetic Systems). During AFM experiments, T cells were maintained at a physiologically relevant temperature 37 °C using a heated stage (Bruker). A soft silicon nitride tipless AFM probe (HQ:CSC38/tipless/Cr-Au, MikroMasch) was used for T cell’s compression. The AFM microcantilevers were pre-calibrated using the standard thermal noise fluctuations method. The estimated spring constants for microcantilevers used were 0.07–0.1 N/m. After calibration, the AFM probe was moved on top of a rounded T cell. Five to ten successive force curves were performed on each T cell. The deflection set-point was set to 20 nm yielding applied forces between 1.5 and 2 nN.
5
Live Cell Nanomechanical Measurements
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Live cell measurements were performed using a Bruker BioScope Catalyst AFM system (Bruker, Santa Barbara, CA) mounted on an inverted Axiovert 200 M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a Confocal Laser Scanning Microscope 510 META (LSM 510 Meta, Carl Zeiss) and a 40× (0.95 NA, Plan-Apochromat) objective lens (Carl Zeiss). A Petri dish heating stage (Bruker) was used to maintain physiological temperature (37 °C) of cells during measurements. Modified AFM microcantilevers with an attached 25 µm-diameter polystyrene microsphere were obtained from Novascan (Novascan, Ames, IA). The AFM probe spring constant was obtained using the thermal tune method built into the AFM system. Calibrated spring constants for the cantilevers ranged from 0.5 to 1 N/m. After cantilever calibration, the AFM probe was placed on top of the nuclear region of an adherent cell. The deflection setpoint was set between 20 and 25 nm, yielding applied forces between 5 and 18 nN. The force curve ramp rate was set to 0.5 Hz and the probe speed ranged between 1.9 and 2.4 µm/s. Multiple consecutive quasi-static force curves were collected on each individual cell with a deflection trigger of 25 nm.
6
Lipid Bilayer Formation and Peroxide Treatment
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20 μL of DPPC, POPC, POPG suspension prepared above were dropped onto the smooth surface of diamond plates, and we incubated them to promote lipid bilayer formation for 2 h in a 60 °C oven. Morphology images were obtained by a BioScope Catalyst AFM system (Bruker Nano, Santa Barbara, CA) with tapping mode in air (DNP-10 tip). Then lipid bilayers were treated with 2% H2O2 solution. Bilayer images were collected again by AFM. All images were analysed using the Nanoscope Analysis 1.8 software, and their roughness were calculated using Root mean square roughness qualification (Rq).
7
AFM Nanoindentation of Live Cells
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AFM nanoindentation was conducted by a BioScope Catalyst AFM system (Bruker Nano, Santa Barbara, CA). In the AFM nanoindentation experiment, contact mode in fluid was applied. DNP-10 tips (Bruker Nano, Santa Barbara, CA) with V-shaped silicon nitride cantilever and pyramidal silicon nitride tip were applied, whereas the actual value of spring constant was calibrated in liquid using the thermal tune method. Hank's balanced salt solution (HBSS) was used as the buffer for force-indentation curve collection. To keep the activity of live cell, each measurement was limited to 1 h after the PDMS substrate was loaded on the AFM stage. Cells were indented with a fixed force of 2 nN and corresponding displacements were recorded. The analysis was performed according to the retrace force-indentation curves. Sneddon model (eqn (1)) was chosen as the fit model to calculate the Young's modulus. In the equation, the half angle (α) of AFM tips was set as 18°while the Poisson ratio (ν) was assumed to be 0.5.
where F is the load force, E is the Young's modulus, and δ is the indentation depth.
For each cell, approximately 25 force-distance curves were collected. At each condition, at least 25 cells were tested. Data analysis was performed by Nanoscope v1.8.
where F is the load force, E is the Young's modulus, and δ is the indentation depth.
For each cell, approximately 25 force-distance curves were collected. At each condition, at least 25 cells were tested. Data analysis was performed by Nanoscope v1.8.
8
Measuring Intracellular Stiffness via AFM
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AFM was used to measure intracellular stiffness as previously described [1, 2, 30] . Briefly, to measure intracellular stiffness, MEFs cultured on soft and stiff polyacrylamide hydrogels in phenol red-free DMEM with 10% FBS were indented with a silicon nitride cantilever (Bruker; spring constant, 0.06 N/m) with a conical AFM tip (40 nm in diameter). AFM in contact mode was applied to single adherent MEFs using a BioScope Catalyst AFM system (Bruker) mounted on a Nikon Eclipse TE 200 inverted microscope. To analyze the stiffness, the first 600 nm of horizontal tip deflection was fit with the Hertz model for a cone. 5 to 10 measurements of intracellular stiffness from each experiment were acquired near the periphery of each cell (8 to 10 cells per experimental condition). AFM curves were quantified and converted to Young's modulus (stiffness) using AFM analysis software (NanoScope Analysis, Bruker).
9
Quantifying Cell Nuclear Mechanics via AFM
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Live cell measurements were performed using a Bruker BioScope Catalyst AFM system (Bruker, Santa Barbara, CA) mounted on an inverted Axiovert 200M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a Confocal Laser Scanning Microscope 510 META (LSM 510 Meta, Carl Zeiss) and a 40x (0.95 NA, Plan-Apochromat) objective lens (Carl Zeiss). A Petri dish heating stage (Bruker) was used to maintain physiological temperature (37 °C) of cells during measurements. Modified AFM microcantilevers with an attached 25 µm-diameter polystyrene microsphere were obtained from Novascan (Novascan, Ames, IA). The AFM probe spring constant was obtained using the thermal tune method built into the AFM system. Calibrated spring constants for the cantilevers ranged from 0.5 N/m to 1 N/m. After cantilever calibration, the AFM probe was placed on top of the nuclear region of an adherent cell. The deflection setpoint was set between 20 nm and 25 nm, yielding applied forces between 5 nN and 18 nN. The force curve ramp rate was set to 0.5 Hz and the probe speed ranged between 1.9 µm/s and 2.4 µm/s. Multiple consecutive quasi-static force curves were collected on each individual cell with a deflection trigger of 25 nm.
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