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35 protocols using snl 10

1

Atomic Force Microscopy Imaging of Hyaluronic Acid

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Atomic force microscope (AFM) imaging was carried out using an Asylum MFP-3D standalone AFM (Oxford Instruments, Santa Barbara, CA, USA). The sharp nitride lever (SNL) tip (SNL-10, a silicon tip with a 0.35 N/m spring constant on a silicon nitride cantilever, Bruker AFM Probes, Camarillo, CA, USA) was used with tapping mode in water or the HA solution at room temperature (as shown in Figure 3).
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2

Structural Analysis of ProMMP-7 Complexes

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ProMMP-7-E195A and truncated forms were prepared at 50 or 100 nM in imaging buffer (10 mM CaCl2, pH 7.5; see SI). Mixtures with Hep dp8 or dp16, heparin, HS, or HS dp8 were examined. Each solution was deposited immediately after mixing on a freshly cleaved muscovite mica surface (V-1 grade, Spi Supplies) and incubated for ~5 min. The surface was then rinsed three times with ~100 µL of imaging buffer. AFM images were acquired in imaging buffer in tapping mode using a commercial instrument (Cypher, Asylum Research). SNL tips (sharp nitride levers, SNL-10, Bruker) with measured spring constants of ~0.2 N/m were used. Images were recorded at ~30°C with an estimated tip-sample force < 100 pN, deduced by comparing the free space tapping amplitude (~5 nm) to the imaging set point amplitude. Under such conditions, minimal protein distortion is expected. Image analysis is detailed in SI.
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3

Atomic Force Microscopy of Test Samples

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Surface morphology of the test samples was studied by the atomic force microscopy on a Multimode microscope with a Nanoscope V controller (Veeco, Plainview, New York, NY, USA). The atomic force microscopy (AFM) observations were performed in air at room temperature under the non-resonance scanning mode (PeakForce Tapping QNM). As probes, the cantilevers based on silicon nitride with a single-crystalline silicon tip SNL-10 (Bruker, Billerica, MA, USA) were used; the nominal resonance frequency was 65 kHz, and the force constant was 0.35 N m−1.
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4

Characterizing Silk Fibroin Substrate Properties

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The surface topography and roughness of the SF substrates were measured using AFM (Bruker, multimode 8, U.S.A.) (n = 3, three random points per sample). The stiffness of the SF films was measured by using AFM cantilevers (SNL-10, Bruker, multimode 8, U.S.A.) with a nominal spring constant of 0.35 N·m−1. Samples for stiffness measurement were first hydrated with PBS solution,38 (link) and the force vs. indentation curves were obtained in PBS on each SF substrate. Elastic modulus values were analyzed by NanoScope Analysis software.
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5

Atomic Force Microscopy of Small EVs

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Images were collected with an Atomic Force Microscope Multimode VIII equipped with a Nanoscope V (Bruker, Santa Barbara, CA, USA) controller and operated in liquid in ScanAsyst mode. The cantilever chosen for imaging was the SNL‐10 (Bruker) with a nominal spring constant of 0.35 N·m−1 and a tip radius of 2 nm. Images were analyzed as described in previous work to describe the sample regarding the size distribution of the small‐EVs and their contact angle, a fingerprint of their mechanical properties [25 (link)].
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6

AFM Imaging of Langmuir-Blodgett Films

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The AFM topographic images of LB films were acquired in air tapping mode using a Multimode AFM controlled by Nanoscope IV electronics (Veeco, Santa Barbara, CA) under ambient conditions. Triangular AFM probes with silicon nitride cantilevers and silicon tips were used (SNL-10, Bruker), which have a nominal spring constant ≈ 0.35 N•m -1 . Images were acquired at 1.5 Hz and at minimum vertical force so as to reduce sample damage. AFM images were obtained from at least two different samples, prepared on different days, and by scanning several macroscopically separated areas on each sample. AFM images have been obtained from LB films transferred at several surface pressures, being the rationale before and after the physical state change and the biologically relevant π = 33 mN•m -1 .
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7

Langmuir-Blodgett AFM Imaging of Interfacial Films

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For AFM imaging, interfacial films were transferred onto a freshly-cleaved mica plate using the Langmuir-Blodgett method. The transfer was processed after 2 hours kinetics at a constant surface pressure and at a very low speed (0.5 mm.min -1 ). Imaging was carried out with an AFM (Multimode Nanoscope 5, Bruker, France) in contact mode QNM in air (20°C), using a standard silicon cantilever (0.06 N/m, SNL-10, Bruker, France), and at a scan rate of 1 Hz. The force was minimized during all scans and the scanner size was 100×100 µm².
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8

Characterizing Mineral-Bacterial Interactions

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The grid index of a single montmorillonite particle, which was later used for cell–mineral interaction against the bacterial coated probe, was recorded using the light microscope integrated in the AFM. Then, the structure of the respective particle was studied in PFQNM mode at 5 nN in air using a new SNL probe (k = 0.12 N m−1, SNL-10, Bruker, USA). The same process was applied to a single R. erythropolis cell before its interaction with the kaolinite probe.
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9

Characterizing Probe Tips for Nanomechanical Mapping

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Before a relocation system with a built-in characterizer could be prepared, the potential characterizers had to be characterized themselves. A detailed description is given in ESI (SI-1, step 1). Briefly, a sharp nitride lever tip (k = 0.12 N m−1, SNL-10, Bruker, USA) was characterized by scanning a titanium roughness sample (RS-12M, Bruker, USA, ESI-Fig. 1a) in Peak Force Quantitative Nanomechanical Mapping (PFQNM) mode in air with an atomic force microscope (AFM, Dimension Icon, Bruker Corporation, USA) and further used as built-in characterizer for the kaolinite modified probe. The titanium roughness sample allows the characterization of the very end of the AFM tip25 (link) which was needed to precisely define the dilation length of the ∼40 nm thick kaolinite sheets. As a characterizer for the bacterial modified tipless probe, the more elongated Tap150A probe (k = 5 N m−1, Bruker, USA) was characterized by tapping mode using a test grating TGT1 (NT-MDT Spectrum Instruments, USA). The TGT1 characterizes the overall tip shape at a sub-micron scale, which was essential for the morphology imaging of the bacteria in μm scale. We always used the frame down command, i.e., a horizontal fast scan direction. The resultant images were flattened by first order and subjected to blind tip reconstruction analysis using NanoScope Analysis software (version 8.15, Bruker).
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10

Atomic Force Microscopy of Exosomes

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Purified exosomes were diluted 1: 50 in deionized water; 5- to 10-μL samples were spotted onto freshly cleaved mica sheets (V-1, thickness 0.15 mm, size 15×15 mm) and dried with mild nitrogen flow for atomic force microscopy (AFM) scanning at room temperature. An Icon AFM (Dimension Icon, Bruker; Camarillo, CA) was used to obtain exosome topographic images at tapping mode, which generates high-resolution images by detecting the change in the vibration amplitude of the cantilever by silicon probes (SNL-10 and Scanasyst-Air, Bruker, USA). A 5×5 μm sample area was scanned in every microscopic field. Topographic images were recorded at 512×512 pixels at a scan rate of 1 Hz and 100 pN force. After that, samples were analyzed in peak force quantitative nanomechanical mapping (Peak Force QNM) mode to obtain nanomechanical maps of exosomes. In Peak Force QNM mode, the silicon probes acted on samples to obtain force-distance curves which are then used as feedback signals to generate the peak force error images. Image processing was performed with NanoScope Analysis software (Bruker; Camarillo, CA).
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