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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.

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3 protocols using atec cont

1

Nanoneedle Fabrication and Functionalization

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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.
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2

Cantilever Probes for Nanodiamond Fixation

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qp-BioAC (tip height 7μ m) and ATEC-CONT (tip height 17μ m) were purchased from Nanosensors and MLCT-BIO-DC (tip height 3.5μ m) was purchased from Bruker. MLCT-BIO-DC-C probes have a resonance frequency of 1 kHz, ATEC-CONT of 6 kHz and qp-BioAC of 25 kHz and 45 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 50 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.
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3

Probing Cell Mechanical Stiffness

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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.
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