Dynamic light scattering (ZetaPALS, Brookhaven Instrument) was used to characterize the colloidal liposomes properties. Size distribution was performed with 5 mM liposomes, diluted 1: 1000 in citric acid/sodium citrate buffer (pH 4) or PBS (pH 7.4). Zeta potential was measured by diluting 5 mM liposomes, 1: 1000 in de-ionized water. To assess morphology, liposomes (2.5 μL at 2 mg/mL) were loaded onto TEM grids (#661-200-CU-100, Ted Pella), cleaned with easiGlow™ (Ted Pella), then cooled in liquid ethane (Vitrobot Mark IV, ThermoFisher). The frozen liposomes were visualized using a cryoEM (TF20 FEI Tecnai-G2, 300 kV in the low dose mode).
Easiglow
Manufactured by Ted Pella
Sourced in United States
The EasiGlow is a laboratory equipment product designed for sputter coating and carbon/metal deposition. It is a compact and easy-to-use system that provides a controlled environment for sample preparation and coating. The EasiGlow is capable of depositing thin films of various materials, including carbon, gold, and other metals, onto sample surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (3 mentions)
Tf20 fei tecnai g2, by Thermo Fisher Scientific (1 mentions)
Titan krios microscope, by Thermo Fisher Scientific (1 mentions)
300mesh grids, by Quantifoil (1 mentions)
Jem 1400plus, by JEOL (1 mentions)
Ht7700, by Hitachi (1 mentions)
A3660, by Merck Group (1 mentions)
Cf 1.2 1 3 4c, by Protochips (1 mentions)
Ultra thin carbon support film, by Agar Scientific (1 mentions)
9 protocols using easiglow
1
Comprehensive Characterization of Colloidal Liposomes
2
Cryo-EM Specimen Preparation for Ribosome Imaging
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The cryo-EM specimens were prepared on QuantiFoil R 2/2 grids with 2-nm continuous carbon (QuantiFoil Micro Tools GmbH, Großlöbichau, Germany). The grids were glow-discharged for 40 s at 20 mA and 0.38 mBar using an EasiGlow (Ted Pella, Inc., Redding, CA, USA). Plunge-freezing was done using a Vitrobot mark IV (Thermo Fisher Scientific, USA) using 3 µL of sample at 160 nM ribosome concentration after 30 s of incubation on the grid at 100% humidity and 10 °C and blotting for 3.5 s. Data were collected on two different occasions on the same Titan Krios microscope from two different grids from the same batch. The microscope was equipped with a Falcon-III direct electron detector used in integrating mode. The nominal magnification was 75,000×, corresponding to a pixel size of 1.09 Å in the specimen plane, and the acceleration tension was 300 kV. On the first occasion, movies were collected with 66.2 e−/pixel/second during 0.773 s for a total dose of 41.0 e−/Å2, and the sample stage was set to either 0° (3052 movies) or 15° (2196 movies) tilt. In the second session, movies were collected with 68.4 e−/pixel/second during 0.770 s for a total dose of 42.2 e−/Å2, and the sample stage was set to a 30° tilt (6120 movies). Data collection parameters are summarized in Table S1 .
3
Cryo-EM Imaging of TbCSV Particles
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Cryo-EM grids were prepared by applying TbCSV particles to 300 mesh grids (Quantifoil Micro Tools GmbH). Grids were glow-discharged for 60 s before applying the sample (easiGlow, Ted Pella). 2.5 μL sample was applied to the grids, blotted immediately using a FEI Vitrobot Mark IV plunge freeze (Thermo Fisher Co) device. Grids were frozen in liquid ethane cooled by liquid nitrogen at 100% relative humidity, and a chamber temperature of 20 °C. TbCSV data were collected on an FEI Titan Krios G2 (Center of Cryo Electron Microscopy, Zhejiang University) EM at 300 kV, using a total electron dose of 40 e-/Å2 and a magnification of 29,000×. A total of 2495 movies were recorded using the SerialEM [16 (link)] semi-automated acquisition software on a Gatan K2 summit direct electron detector in counting mode, with a final pixel size of 1.014 Å/pixel. Each exposure movie had a total exposure of 5 s and contained 20 frames.
4
Negative Staining of Extracellular Vesicles
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Negative staining was performed at the Electron Microscopy Unit (EMU) of the Imaging Center Essen (IMCES) for HEK293T and HeLa TSU and PSU sEV fractions that have more purity (F2 and F3) along with F4 that has less purity. In addition, FBS18 sEV fractions were included as a negative control. Briefly, 3 µL of sEV fractions (F2–F4) was added onto a Formvar- and carbon-coated 200 mesh copper grid (#S162, PLANO GmbH, Wetzlar, Germany) which had a hydrophilic surface due to being exposed to glow discharging for 30 s at 15 mA (easiGlow™, TedPella Inc., Redding, CA, USA). Samples were then negatively stained by placing the grid on the top of the droplet of 1.5% aqueous Phosphotungstic acid solution (w/v, 2635.1, Carl Roth, Karlsruhe, Germany) for a minute. Excess liquid was removed using filter paper, and the grids were allowed to dry for at least five minutes under ambient air. Images were acquired using JEOL JEM 1400Plus (JEOL Ltd., Tokyo, Japan) operating at 120 kV and with a 4096 × 4096 pixels CMOS camera (TVIPS, Gauting, Germany). Image acquisition software EMMENU (version 4.09.83) was used for taking 16-bit images. Image post-processing and analysis were carried out using ImageJ (version 1.52a) to determine the average diameter of sEVs.
5
Transmission Electron Microscopy of Cells
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For electron microscopy experiment, all cells were prepared by the ChromEM staining protocol and embedded in Durcupan resin (EMS) (27 (link)). After curing, 40-nm-thin sections were made and deposited onto copper 200-mesh grid with carbon/formvar film (EMS). The grids were plasma-cleaned by a plasma cleaner (easiGlow, TED PELLA) before use. An HT7700 (HITACHI) transmission electron microscopy (TEM) was used to record TEM images of cell sections at 80 kV with a pixel size of 2.5 nm.
6
Apoferritin Samples for Cryo-EM Imaging
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Apoferritin samples were prepared to a final protein concentration of 0.25 mg/mL in PBS from horse-spleen apoferritin (#A3660, lot #SLBF8335V, Sigma-Aldrich, St. Louis, MO) and contained 15 nm gold particles (#1115, Nanoprobes, Yaphank, NY). A 3 µL aliquot of the solution was applied to a freshly glow-discharged (60 s. at 15 mA, Easiglow, Ted Pella, Inc., Redding, CA) holey carbon support grid (#CF-1.2/1.3–4C, Protochips, Morrisville, NC) and plunge frozen using a Vitrobot Mark two (FEI; 5 s. blotting time, blotting force 5, 85% relative humidity). The sample was then transferred into liquid nitrogen and stored until imaging.
7
Cryo-EM Imaging of Purified Ribosomes
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400 mesh copper grids with a supporting carbon lacey film coated with an ultra-thin carbon support film < 3 nm thick (Agar Scientific, UK) were employed. Grids were glowdischarged for 30 seconds (easiGlow, Ted Pella) prior to applying 3 µL of purified ribosomes, and vitrification was performed by plunge-freezing in liquid ethane cooled by liquid nitrogen using a Leica EM GP device (Leica Microsystems). Samples were diluted using the buffer exchange buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM MgCl2) as required. Cryo-EM data was collected on a FEI Titan Krios (Astbury Biostructure Laboratory, University of Leeds) EM at 300 kV, using a total electron dose of 80 e -/Å 2 and a magnification of 75,000 × at -2 to -4 μm defocus. Movies were recorded using the EPU automated acquisition software on a FEI Falcon III direct electron detector, in linear mode, with a final pixel size of 1.065 Å/pixel (Sup Table 2).
8
Cryo-EM Visualization of PCV-1 VLPs
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Cryo-EM grids were first glow discharged for 30 seconds (easiGlow, www.tedpella.com).
Samples were prepared in a chamber held at 95-100% relative humidity and 4 °C. 3 ul of purified PCV-1 VLPs were applied to 400 mesh lacey grids with an ultra-thin carbon support.
The sample was left to incubate on the grid for 1 minute and blotted using a FEI vitrobot mark IV (www.thermofisher.com). Grids were vitrified in liquid ethane cooled by liquid nitrogen. Data was collected on a ThermoFisher Titan Krios electron microscope (Astbury Biostructure Laboratory, University of Leeds) at 300 kV. Images were taken at 75,000x magnification, with a total electron dose of 66.1 e-/Å 2 . Exposures were recorded using the EPU automated acquisition software on a Falcon III detector. Movies had a total exposure time of 1.5 seconds split across 59 fractions. Detailed parameters for data collection are shown in Table S1. Data collection protocol was derived from that described in Thompson et al 2019 (Thompson et al., 2019) .
Samples were prepared in a chamber held at 95-100% relative humidity and 4 °C. 3 ul of purified PCV-1 VLPs were applied to 400 mesh lacey grids with an ultra-thin carbon support.
The sample was left to incubate on the grid for 1 minute and blotted using a FEI vitrobot mark IV (www.thermofisher.com). Grids were vitrified in liquid ethane cooled by liquid nitrogen. Data was collected on a ThermoFisher Titan Krios electron microscope (Astbury Biostructure Laboratory, University of Leeds) at 300 kV. Images were taken at 75,000x magnification, with a total electron dose of 66.1 e-/Å 2 . Exposures were recorded using the EPU automated acquisition software on a Falcon III detector. Movies had a total exposure time of 1.5 seconds split across 59 fractions. Detailed parameters for data collection are shown in Table S1. Data collection protocol was derived from that described in Thompson et al 2019 (Thompson et al., 2019) .
9
TEM Imaging of Nanoparticle Samples
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TEM images of NPs were acquired using a JEOL electron microscope (JEM 100 CX II, operating at 120 kV) equipped with an US1000 2k × 2k camera (Gatan). To do so, 5 μL of samples were deposited onto a 400 mesh carbon-coated copper grid beforehand treated with a glow discharge (easiGlow, Ted Pella Inc).
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