Nanoneedles were fabricated from pyramidal silicon AFM cantilevers (ATEC-Cont, Nanosensors) and etched to a cylindrical shape of 200 nm in diameter and 10-15 µm in length using the FIB 31 (link). Spring constants (k = 0.1-0.4 N/m) were determined as described above. The silicon surface was cleaned with oxygen plasma in a JPA300 plasma asher (200 W, 5 min) (J-science, Kyoto, Japan) and treated with 1% HF for 1 min. After repeating the plasma (10 min) and 1% HF treatment, the nanoneedle was modified by physical adsorption of 50 μg/ml of ZZ-BNC at room temperature for 1 h; ZZ-BNC is bio-nanocapsule-based anchor to which the Fc domain of antibodies can bind 40 (link). Anti-vimentin (V6630; Sigma-Aldrich) or anti-nestin antibodies (SAB4200347; Sigma-Aldrich) (11.75 µg/ml each) were bound to the ZZ-domain of the ZZ-BNC by incubating in PBS overnight at 4°C. The antibody-immobilized nanoneedle was rinsed prior to use in cell fishing experiments. Force measurements were carried out using the AFM Nanowizard II with CellHesion. For cellular membrane penetration tests, nanoneedles were inserted into cells at an approach velocity of 10 µm/s with a set point of 200 nN, left to dwell within the cells for 60 s, and then evacuated at 10 µm/s. Ten different sites on each cell were targeted for insertion and three cells were tested for each cell type.
Atec cont
Manufactured by Nanosensors
Sourced in Switzerland
The ATEC-Cont is a compact and versatile laboratory equipment designed for continuous monitoring applications. It provides reliable and accurate measurements of various parameters in real-time. The core function of the ATEC-Cont is to enable continuous data acquisition and monitoring for research and analysis purposes.
Automatically generated - may contain errors
3 protocols using atec cont
1
Nanoneedle Fabrication and Functionalization
2
Cantilever Probes for Nanodiamond Fixation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qp-BioAC (tip height m) and ATEC-CONT (tip height m) were purchased from Nanosensors and MLCT-BIO-DC (tip height m) was purchased from Bruker. MLCT-BIO-DC-C probes have a resonance frequency of kHz, ATEC-CONT of kHz and qp-BioAC of kHz and kHz (CB2 and CB1, respectively), all resonances measured in liquid environment. Custom tips, presenting a plateau at the tip apex, were purchased from Nanoworld: they were fabricated modifying a qp-BioAC cantilever (see Section 4 of Supplementary Information ). An electron beam deposited carbon tip was grown on top of a thin layer of gold. Subsequently, a flat circular plateau of nm diameter was obtained at the tip apex by means of Field Ion Beam (FIB) to aid fluorescent nanodiamond fixation and stability.
For optomechanical force characterization, a dozen tips were used to fully characterize and understand the forces employed. All probes of the same brand behaved similarly, with the same range of forces and same force/intensity distribution over the cantilever. For the approach-retract experiment with the nanodiamonds, we have collected data from tens of different probes whereas each probe had a different nanodiamond that was carefully grafted. In Fig.6 two of such different tips were used.
For optomechanical force characterization, a dozen tips were used to fully characterize and understand the forces employed. All probes of the same brand behaved similarly, with the same range of forces and same force/intensity distribution over the cantilever. For the approach-retract experiment with the nanodiamonds, we have collected data from tens of different probes whereas each probe had a different nanodiamond that was carefully grafted. In Fig.
3
Probing Cell Mechanical Stiffness
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the estimation of membrane stiffness of cells expressing fusion proteins, we used atomic force microscopy (AFM; Nanowizard II BioAFM; JPK Instruments, Berlin, Germany), combined with an inverted microscope (Olympus). A normal pyramidal AFM tip (ATEC-Cont, Nanosensors, Neuchatel, Switzerland) was etched to form a cylinder-shape AFM tip, 2.5 μm in diameter and 7.0 μm in height, using a focused ion beam (SMI500, Hitachi High-Tech Science, Tokyo, Japan), which was then used for the determination of mechanical stiffness. U2OS cells were cultured on a plastic dish and transfected with expression vectors for each actin probe. Transformants exhibiting green fluorescence were indented with the cylindrical AFM tip at an approach velocity of 10 μm/s, avoiding the nuclear region, until a repulsive force of 10 nN was reached. The resulting force-indentation curves were fitted by the following equation of the Hertz model for the cylindrical tip:
where F, a, E, I, and ν represent force, the radius of the cylinder, Young's modulus, the indentation depth, and Poisson's ratio; we set at 0.5 in this study referring to a previous study (Chiou et al., 2013) , to evaluate Young's moduli of the tested cells as parameters of cell stiffness (Obataya et al., 2005) . We used the Student's t-test comparing between control cells (EGFP) and each cell line expressing GFP tags for actin.
where F, a, E, I, and ν represent force, the radius of the cylinder, Young's modulus, the indentation depth, and Poisson's ratio; we set at 0.5 in this study referring to a previous study (Chiou et al., 2013) , to evaluate Young's moduli of the tested cells as parameters of cell stiffness (Obataya et al., 2005) . We used the Student's t-test comparing between control cells (EGFP) and each cell line expressing GFP tags for actin.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!