Mlct probe

Manufactured by Bruker

Sourced in United States

The MLCT probe is a type of atomic force microscopy (AFM) probe designed for Bruker's line of AFM systems. It is used for measuring the topography and physical properties of sample surfaces at the nanoscale. The MLCT probe features a silicon nitride cantilever with an integrated tip for high-resolution imaging and force measurements.

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Lab products found in correlation

Nanowizard 3, by Bruker (1 mentions) Axio observer d1, by Zeiss (1 mentions) Mfp3d bio system, by Oxford Instruments (1 mentions) Multimode 8 afm, by Bruker (1 mentions) Mfp 3d bio afm, by Oxford Instruments (1 mentions) Igor pro 6.34a, by Oxford Instruments (1 mentions)

8 protocols using mlct probe

Determining Cell Stiffness via AFM

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Cell stiffness was determined with atomic force microscopy (AFM) Park XE-100 and analyzed with SPIP software. After 7 days cells cultured in static and flow conditions samples with live cells were analyzed with MLCT probes (Bruker Inc.) in liquid environment.

Calibasi Kocal G., Güven S., Foygel K., Goldman A., Chen P., Sengupta S., Paulmurugan R., Baskin Y, & Demirci U. (2016). Dynamic Microenvironment Induces Phenotypic Plasticity of Esophageal Cancer Cells Under Flow. Scientific Reports, 6, 38221.

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Force Spectroscopy of DSG2 Binding in Cardiac Myocytes

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Force spectroscopy on living HL-1 cardiac myocytes was performed as described previously [35 (link)]. Briefly, eukaryotically expressed DSG2-Fc protein was coupled to Si3N4 AFM cantilevers (MLCT probes, Bruker, Calle Tecate, CA, USA) in a concentration of 0.15 mg/ml via a bifunctional polyethylene glycol spacer (acetal-PEG-NHS, Gruber Lab, Institute of Biophysics, Linz, Austria). Experiments were performed with the pyramidal-shaped D-tip (nominal spring constant: 0.03 N/m) clamped into a Nanowizard III atomic force microscope (JPK instruments, Berlin, Germany) mounted on an optical fluorescence microscope (Axio Observer D1, Carl Zeiss) at 37 °C. SPM Control v.4 software (JPK instruments) was used for data acquisition. Force measurements were performed in a region of 5 μm × 1.25 μm spanning the cell–cell contact area of HL-1 cells with following settings: 64 × 16 pixel grid, setpoint 0.2 nN, extend speed 5 μm/s, extend delay 0.1 s. To determine the binding force between the tip-coupled DSG2 and the proteins on the cell surface, the functionalized tip was repeatedly approached to and retracted from the surface. If binding between the tip and surface occurred, the force necessary to rupture the binding (termed as “unbinding force”) was detected by determination of the cantilever’s deflection. The same cell–cell border was compared before and after addition of digitoxin.

Schinner C., Olivares-Florez S., Schlipp A., Trenz S., Feinendegen M., Flaswinkel H., Kempf E., Egu D.T., Yeruva S, & Waschke J. (2020). The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner. Basic Research in Cardiology, 115(4), 46.

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AFM Imaging of Native Cells

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To study fixed preparations, we used a sample nearly identical to the SEM method with glass slides serving as a substrate. The cells were scanned in a semi-contact mode in the air by Ntegra Spectra system (NT-MDT, Russia). To process the scanning results, we used the Topometrix software package (USA). The test involved the use of DNP probes (Bruker, USA) with a tip radius of 20 nm, front angle of 15°, resonance frequency 65 kHz, spring constant of 0.35 N/m.
The real-time AFM studies of native cells were carried out using the method described in a previous study (Pleskova et al., 2005 (link)). The morphology was studied in the vital state. The test involved the use of MLCT probes (Bruker, USA) with the tip radius of 20 nm, front angle 15°, and resonance frequency in fluid 14 kHz. After 30 min of control scanning, the QDs were added into the Petri dish in CL₅₀ concentration (final concentration 0.025 mg/ml and 0.04 mg/ml for CdSe/ZnS-MPA and CdSe/CdZnS/ZnS-PTVР respectively).

Pleskova S.N., Kriukov R.N., Gorshkova E.N, & Boryakov A.V. (2019). Characteristics of quantum dots phagocytosis by neutrophil granulocytes. Heliyon, 5(3), e01439.

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Calibrating Atomic Force Microscope Probes

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Cantilever A was kept and the other five cantilevers of the MLCT probe (Bruker) were knocked out. All the experiments were performed in PBS solution. The fused silica was used to calibrate the probe's deflection sensitivity. Briefly, the force‐indentation curves were obtained on a relative hard sample based on contact mode. Afterward, the contact area between the probe and the sample within the force curve was selected to calculate the reflection sensitivity. The thermal tune mode was used to calibrate the K (elastic coefficient) of the probe. Then the thermal vibration energy of the cantilever was analyzed by a fitting curve. After obtaining deflection sensitivity and K, a complete picture of the standard roughness sample (RS‐15M) was captured using ScanAsyst mode to correct tip radius.

Ye S., Li W., Wang H., Zhu L., Wang C, & Yang Y. (2021). Quantitative Nanomechanical Analysis of Small Extracellular Vesicles for Tumor Malignancy Indication. Advanced Science, 8(18), 2100825.

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Atomic Force Microscopy for Cell Elasticity

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AFM is performed using the MFP-3D-BIO system (Asylum Research, Oxford
Instruments). Cells are probed with the “C” tip of an MLCT probe
(Bruker) at room temperature. The sensitivity and spring constant of each probe
(11.5 to 14.5 mN/m) are calibrated before each experiment. Cells are plated on a
polystyrene petri dish coated with a thin layer of Matrigel (100 μg/ml)
approximately 24 hours prior to each experiment. Force curves are acquired by
indenting the central cytoplasmic region of 25 to 35 cells for each cell line.
To avoid possible contribution of adjacent cells, only single cells were probed.
Approach and retract speeds for all experiments are 5 μm/s. The elastic
modulus for each cell is determined by fitting force curves to the Hertz-Sneddon
model^{87 ,88 (link)} using Asylum Research software.

Nguyen A.V., Nyberg K.D., Scott M.B., Welsh A.M., Nguyen A.H., Wu N., Hohlbauch S.V., Geisse N.A., Gibb E.A., Robertson A.G., Donahue T.R, & Rowat A.C. (2016). Stiffness of pancreatic cancer cells is associated with increased invasive potential. Integrative biology : quantitative biosciences from nano to macro, 8(12), 1232-1245.

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Atomic Force Microscopy of RBC Cytoskeleton

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JPK Nanowizard I AFM (JPK Instrument AG, Germany) was used to image both the smeared samples and cytoplasmic-surface exposed samples. Smear samples were imaged in air contact mode using MLCT probe (Bruker, Billerica, MA, USA). Super sharp silicon probes (SSS-NCHR probes, Nanosensor, Neuchatel, Switzerland) with tip radius of about 2 nm were used to image RBC cytoskeleton in tapping mode9 (link). Height images were captured at a resolution of 256 × 256 pixels for 10 µm × 10 µm or 512 × 512 pixels for 1 µm × 1 µm areas. Individual spectrin filaments and mesh were analyzed using ImageJ for estimating the average lengths and sizes9 (link).

Sinha A., Chu T.T., Dao M, & Chandramohanadas R. (2015). Single-cell evaluation of red blood cell bio-mechanical and nano-structural alterations upon chemically induced oxidative stress. Scientific Reports, 5, 9768.

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AFM Analysis of Nanogel Mechanical Properties

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AFM force measurements were conducted using a MultiMode 8 AFM (Bruker) with an inverted optical microscope. The nanogels in 1 × PBS dispersion with or without light irradiation were dropped onto a monocrystalline silicon substrate. MLCT probe (Bruker) and cantilever A were used (f₀ = 22 kHz, k = 0.07 N m⁻¹). The force curves were measured in shooting mode, with the Z piezo working in a closed loop. Young’s modulus of the nanogels was calculated by applying the Sneddon-modified Hertz model.

Zhang D., Tian S., Liu Y., Zheng M., Yang X., Zou Y., Shi B, & Luo L. (2022). Near infrared-activatable biomimetic nanogels enabling deep tumor drug penetration inhibit orthotopic glioblastoma. Nature Communications, 13, 6835.

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AFM-Based Quantification of Cellular Stiffness

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An MFP-3D-Bio AFM (Asylum Research, Santa Barbara, CA) was used to take 90 × 90 μm force maps (Fig. 2a–d) of selected regions of interest within each sample. Each force map includes a 32×32 grid of separate force curves which were each acquired using a trigger point of 200 pN and indenting 250 nm at a rate of 600 nm s⁻¹. Force curves can be fitted with different theoretical models in order to accurately estimate the elastic modulus of cell components²³. Thus, in order to calculate cortical stiffness, the first 100 nm of indentation in the force curves were fitted with the Hertz-Sneddon model with a pyramidal indenter³³. Data was collected on a minimum of 12 cells per condition for each experiment, as each ROI contained at least three cells from the monolayer. In addition to determining stiffness distributions of these monolayers, AFM imaging (Fig. 3a–d) was performed. 90×90 μm images were taken at .10Hz with a spacing of 0.352 μm between lines. All stiffness measurements were taken with TR400PB silicon nitride pyramidal tips with a height of 3 μm and a semi-included angle of 35 degrees (Olympus, Tokyo, Japan), and AFM images were taken using a MLCT probe (Bruker, Billerica, MA). Asylum Research’s software (Igor Pro 6.34A) was used for thermal tune calibration of the cantilever spring constant.

Wong A.K., LLanos P., Boroda N., Rosenberg S.R, & Rabbany S.Y. (2015). A Parallel-Plate Flow Chamber for Mechanical Characterization of Endothelial Cells Exposed to Laminar Shear Stress. Cellular and molecular bioengineering, 9(1), 127-138.

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