Six microliters of each sample was placed onto Lacey Carbon 300 Mesh Copper grids (Ted Pella, Redding, California) and allowed to sit for 2 min, with the excess wiped off by a Kimwipe. Stir-20h and bottom samples were diluted 100x with A5 buffer prior to being loaded onto the TEM grids. FACS sorted samples, heat-stressed, unstressed control, and buffers were loaded directly onto the TEM grids with no dilution. The grids were then placed into filtered 2% uranyl acetate for 2 min and extra stain was removed with a Kimwipe. The wet grids were air-dried for several min prior to being examined by TEM. Samples were imaged on an FEI Technai F20 XT Field Emission Transmission Electron Microscope (Hillsboro, OR) using 200 kV electron acceleration voltage. Images were captured at a standardized, normative electron dose and at a constant defocus value from the carbon-coated surfaces.52 (link)
Lacey carbon 300 mesh copper grid
Manufactured by Ted Pella
Sourced in United States
The Lacey Carbon 300 mesh copper grid is a laboratory tool commonly used for specimen preparation in electron microscopy. It provides a stable support structure for thin samples, allowing for effective imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (3 mentions)
Vitrobot mark 3, by Thermo Fisher Scientific (3 mentions)
Whatman, by Cytiva (2 mentions)
Cryo holder system, by Ametek (2 mentions)
4096 pixel ccd eagle camera, by Thermo Fisher Scientific (2 mentions)
Tecnai g2 20 transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Eagle camera 4x4 k, by Thermo Fisher Scientific (2 mentions)
Succinic acid, by Merck Group (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Avantor (1 mentions)
Nitric acid, by Avantor (1 mentions)
Sodium azide, by Avantor (1 mentions)
Ammonium nitrate, by Thermo Fisher Scientific (1 mentions)
Citric acid monohydrate, by Thermo Fisher Scientific (1 mentions)
Tartronic acid, by Merck Group (1 mentions)
Glutaric acid, by Thermo Fisher Scientific (1 mentions)
Sodium nitrate, by Avantor (1 mentions)
Pimelic acid, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Tricarballylic acid, by Merck Group (1 mentions)
12 protocols using lacey carbon 300 mesh copper grid
1
Transmission Electron Microscopy Imaging
2
Characterization of Organic Acids and Compounds
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The following chemicals, including their sources, purity, and CAS numbers, were used: citric acid monohydrate, Fisher, ACS grade, 5949-29-1; ᴅʟ-malic acid, Alfa Aesar, 98%, 6915-15-7; ᴅʟ-isocitric acid, trisodium salt hydrate, Acros Organics, 95%, 1637-73-6; ᴅʟ-lactic acid, TCI, >85%, 50-21-5; ᴅʟ-glyceric acid, TCI, 20% in water (ca. 2 mol/L), 473-81-4; ᴅʟ-tartaric acid, TCI, >99%, 133-37-9; glutaric acid, Acros Organics, 99% 110-94-1; tricarballylic acid, Alfa Aesar, 98%, 99-14-9; adipic acid, Sigma, 99%, 124-04-9; acetic acid, Sigma, ACS grade, 64-19-7; pimelic acid, Alfa Aesar; >98%, 111-16-0; succinic acid, TCI, >99%, 110-15-6; tartronic acid, Sigma, >97%, 80-69-3; sodium mesoxalate monohydrate, Chemodex, >98%, 31635-99-1; ᴅʟ-2-hydroxybutyric acid sodium salt, Alfa Aesar, >97%, 5094-24-6; ᴅʟ-3-hydroxybutyric acid sodium salt, Chem Impex Int’l Inc., 100.3%, 150-83-4/306-31-0; ascorbic acid, TCI, >99%, 50-81-7; ammonium nitrate, Fisher, ACS grade, 6484-52-2; sodium nitrate, VWR, ACS grade, 7631-99-4; sodium hydroxide, VWR, ACS grade, 1310-73-2; nitric acid, Sigma, ACS grade, 7697-37-2; and sodium azide, Sigma, 99.8%, 26628-22-8. Lacey carbon, 300 mesh, copper grids (product #01895) from Ted Pella, Inc. were used for electron microscopy.
3
Cryo-EM Sample Preparation Protocol
4
Cryo-EM Imaging Using Lacey Carbon Grids
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Lacey carbon 300 mesh copper
grids (Ted Pella) were used for cryo-EM imaging on FEI TALOS F200C
with a Ceta 16 M camera at 200 kV accelerating voltage. PELCO easiGlow
Glow Discharge Cleaning System was also used for the plasma treatment
of the grids (Supporting Information ).
grids (Ted Pella) were used for cryo-EM imaging on FEI TALOS F200C
with a Ceta 16 M camera at 200 kV accelerating voltage. PELCO easiGlow
Glow Discharge Cleaning System was also used for the plasma treatment
of the grids (
5
Cryo-EM Analysis of Actin-C2 Interaction
6
Cryo-TEM Analysis of Extracellular Vesicles
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Cryo-TEM analysis was carried out with fresh DC-derived EVs samples resuspended in 0.1 M phosphate buffer (pH 7.2) in the Electronic Cryo-Microscopy Unit of the Scientific and Technological Centres of the University of Barcelona (CCiT-UB, Barcelona, Spain). Each EV suspension (5 μL) was placed on the carbon surface of a glow-discharged Lacey Carbon 300 mesh copper grid (Ted Pella, Redding, CA, USA). The sample was allowed to adsorb for 4 min at 100% humidity inside the chamber of a Vitrobot Mark III (FEI Company, Eindhoven, Netherlands). Excess liquid was blotted with filter paper, followed by cryo-immobilization by plunge freezing in liquefied ethane. The vitrified sample was stored in liquid nitrogen until its observation. Plunge-frozen samples were transferred to a Tecnai F20 microscope (FEI, Eindhoven, The Netherlands) using a cryo-holder system (Gatan, Pleasanton, CA, USA). The sample was examined at 200 kV, at a temperature ranging from −179 to −170 °C, using low-dose imaging conditions. Images were recorded with a 4096 × 4096-pixel CCD Eagle camera (FEI, Eindhoven, The Netherlands).
7
Cryo-TEM Analysis of Extracellular Vesicles
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Cryo-transmission electron microscopy (Cryo-TEM) analysis was performed with fresh (4 °C stored) EVs samples as described previously38 (link). Briefly, one drop of the sample diluted 1/5 in milliQ water was applied on the carbon surface of a glow-discharged Lacey Carbon 300 mesh copper grid (Ted Pella, USA). The sample was allowed to adsorb for 1 min, at 100% humidity, inside the chamber Vitrobot Mark III (FEI Company, Eindhoven, Netherlands). The excess of liquid was automatically blotted with filter paper, followed by cryo-immobilization by plunge freezing in liquefied ethane. The vitrified sample was stored in liquid nitrogen until its observation in the cryo-TEM microscope. Plunge-frozen sample was transferred to a Tecnai F20 EM (FEI Companys) using a cryo-holder system (Gatan, Pleasanton, USA). The sample was examined at 200 kV, at a temperature ranging from − 179 to − 170 °C, using low-dose imaging conditions. Low-dose images were recorded with a 4096 × 4096-pixel CCD Eagle camera (FEI Company). This analysis was carried out at the Cryo-Electron Microscopy Unit of the Science and Technological Centres at the University of Barcelona (CCiT-UB).
8
Nanodispersion Morphological Characterization
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The morphological characterization of the selected TQ-free and TQ-loaded nanodispersions was performed, as described in our recent reports [34 (link),35 (link)]. Briefly, 3–4 µL of the nanodispersion was applied on a hydrophilized lacey carbon 300 mesh copper grid (Ted Pella Inc., Redding, CA, USA). The observations were done with Tecnai G2 20 transmission electron microscope (FEI, Eindhoven, Holland) at a voltage of 200 kV under a low-dose rate (~5 e/Å2s). Images were then recorded using a FEI Eagle camera 4k × 4k at a nominal magnification of 69,000× resulting in a final image sampling of 0.22 nm/pixel.
9
Cryo-TEM Imaging of CNC-g-POx Dispersions
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TEM images were
recorded in low-dose conditions using a TALOS F200C (Thermo Fischer
Scientific) microscope working at 80 kV equipped with a Ceta 16 M
4 k × 4 k pixel CMOS camera (Thermo Fischer Scientific). TEM
samples were prepared by the deposition of CNC-g-POx
dispersions (0.01 wt %) on a carbon 400 mesh copper grid (Ted Pella
Inc.) and removing the excess after 1 min, followed by staining with
a 1% uranyl acetate solution for 30 s.
Cryo-TEM images were
recorded in low-dose conditions, with a defocus range of 1–3
μm, using a TALOS F200C (Thermo Fischer Scientific) microscope
working at 80 kV equipped with a Ceta 16 M 4 k × 4 k pixel CMOS
camera (Thermo Fischer Scientific). Samples were prepared by deposition
of CNC-g-POx dispersions (0.1 wt %) onto a Lacey
Carbon 300 mesh copper grid (Ted Pella Inc.) in a controlled environment
vitrification system (Vitrobot Mark IV, Thermo Fischer Scientific—formerly
FEI) at controlled temperature (22 °C) in 100% humidity, blot
force of 5 and blot time of 2.5 s.
For both sample preparations,
grids were previously subjected to
a glow discharge treatment using an easiGlow discharge system (Pelco)
with 15 mA negative current for 10 s in air atmosphere to make them
hydrophilic. Sample preparation and data acquisition were performed
at the Electron Microscopy Laboratory/Brazilian Nanotechnology National
Laboratory (LNNano).
recorded in low-dose conditions using a TALOS F200C (Thermo Fischer
Scientific) microscope working at 80 kV equipped with a Ceta 16 M
4 k × 4 k pixel CMOS camera (Thermo Fischer Scientific). TEM
samples were prepared by the deposition of CNC-g-POx
dispersions (0.01 wt %) on a carbon 400 mesh copper grid (Ted Pella
Inc.) and removing the excess after 1 min, followed by staining with
a 1% uranyl acetate solution for 30 s.
Cryo-TEM images were
recorded in low-dose conditions, with a defocus range of 1–3
μm, using a TALOS F200C (Thermo Fischer Scientific) microscope
working at 80 kV equipped with a Ceta 16 M 4 k × 4 k pixel CMOS
camera (Thermo Fischer Scientific). Samples were prepared by deposition
of CNC-g-POx dispersions (0.1 wt %) onto a Lacey
Carbon 300 mesh copper grid (Ted Pella Inc.) in a controlled environment
vitrification system (Vitrobot Mark IV, Thermo Fischer Scientific—formerly
FEI) at controlled temperature (22 °C) in 100% humidity, blot
force of 5 and blot time of 2.5 s.
For both sample preparations,
grids were previously subjected to
a glow discharge treatment using an easiGlow discharge system (Pelco)
with 15 mA negative current for 10 s in air atmosphere to make them
hydrophilic. Sample preparation and data acquisition were performed
at the Electron Microscopy Laboratory/Brazilian Nanotechnology National
Laboratory (LNNano).
10
Cryo-EM Visualization of Microvesicles
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The MVs were cryoimobilized using the Plunge Freezing technique (Delgado et al., 2019 (link)) for Cryo-EM visualization. The cryo-immobilization was performed in the Vitrobot Mark III (FEI, Eindhoven, Netherlands). One 3μl drop of the suspension was applied on the carbon surface of a glow-discharged Lacey Carbon 300 mesh copper grid (Ted Pella, United States) and held for 1–4min at 100% humidity. The excess liquid was automatically blotted with filter paper, and the sample was immediately plunge-frozen in liquefied ethane. The vitrified sample was then transferred to a Tecnai F20 EM (FEI, Eindhoven, Netherlands) using a cryo-holder (Gatan, Pleasanton, United States). The visualization of samples was carried out at 200kV, at temperatures between −180 and−170°C and at low-dose image conditions. The images were acquired with a 4,096×4,096-pixel CCD Eagle camera (FEI, Eindhoven, Netherlands). The quantification and subsequent analyses of the different MVs were carried out with the ImageJ program (Schindelin et al., 2012 (link)).
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