Purified Fabs (C002, C119, and C121) were complexed with SARS-CoV-2 S trimer at a 2-fold molar excess for 1 min and diluted to 40 μg/mL in TBS immediately before adding 3 μL to a freshly-glow discharged ultrathin, 400 mesh carbon-coated copper grid (Ted Pella, Inc.). Samples were blotted after a 1 min incubation period and stained with 1% uranyl formate for an additional minute before imaging. Micrographs were recorded on a Thermo Fisher Talos Arctica transmission electron microscope operating at 200 keV using a K3 direct electron detector (Gatan, Inc) and SerialEM automated image acquisition software43 (link). Images were acquired at a nominal magnification of 28,000x (1.44 Å/pixel size) and a −1.5 to −2.0 μm defocus range. Images were processed in cryoSPARC, and reference-free particle picking was completed using a gaussian blob picker44 (link). Reference-free 2D class averages and ab initio volumes were generated in cryoSPARC, and subsequently 3D-classified to identify classes of S-Fab complexes, that were then homogenously refined. Figures were prepared using UCSF Chimera45 (link). The resolutions of the final single particle reconstructions were ~17–20 Å calculated using a gold-standard FSC (0.143 cutoff) and ~24–28 Å using a 0.5 cutoff.
400 mesh carbon coated copper grid
Manufactured by Ted Pella
Sourced in United States
The 400 mesh carbon-coated copper grid is a type of lab equipment used in electron microscopy. It is designed to support and hold samples for examination under an electron microscope. The grid features a mesh pattern with 400 squares per inch, and the surface is coated with a thin layer of carbon to provide a conductive surface for the sample.
Automatically generated - may contain errors
Lab products found in correlation
Talos arctica, by Thermo Fisher Scientific (3 mentions)
K3 direct electron detector, by Ametek (3 mentions)
Tecnai g2 spirit biotwin microscope, by Thermo Fisher Scientific (2 mentions)
Dmpc lipids, by Avanti Polar Lipids (2 mentions)
Bio beads sm 2, by Bio-Rad (2 mentions)
Jem 1400plus, by JEOL (2 mentions)
Jem 2010 tem, by JEOL (1 mentions)
Eagle 4k 4k ccd camera, by Thermo Fisher Scientific (1 mentions)
Fei tecnai g20 electron microscope, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Infinite 200 pro plate, by Tecan (1 mentions)
Dynapro plate reader 2, by Wyatt Technology (1 mentions)
Megaview 3, by Olympus (1 mentions)
Jem 1010, by JEOL (1 mentions)
Pelco easiglow system, by Ted Pella (1 mentions)
Mastercycler gradient thermocycler, by Eppendorf (1 mentions)
H 7650 tem, by Hitachi (1 mentions)
Milli q ultrapure water, by Merck Group (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Jem 1400, by JEOL (1 mentions)
22 protocols using 400 mesh carbon coated copper grid
1
Cryo-EM Visualization of SARS-CoV-2 S-Fab Complexes
2
TEM Analysis of Nanoparticle Morphology
3
Negative Stain Electron Microscopy of XFS
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Negative stain electron microscopy was used to assess the different fibrillar deposits of patient-derived XFS samples. XFS materials were removed from the anterior lens capsule surface as described above in sample collection section. XFS patients were classified as having low, moderate, or heavy deposits. 6 μl of each sample was loaded onto a glow-discharged 400 mesh carbon-coated copper grid (Ted Pella Inc. Redding, CA). Typically, samples were adsorbed onto the grid surface for 2 min after which excess solution was removed by blotting with Whatman paper. Grids were then washed briefly and negatively stained using freshly filtered 2% uranyl acetate (UA) or 2% phosphotungstic acid (PTA) solutions. The negatively stained samples were analyzed with a FEI Tecnai G20 electron microscope (FEI Company). An acceleration voltage of 200 kV was used to record micrographs on an Eagle 4 k × 4 k CCD camera (FEI Company).
4
Cryo-EM Analysis of SARS-CoV-2 S-Fab Complexes
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Purified Fabs (C002, C119, and C121) were complexed with SARS-CoV-2 S trimer at a 2-fold molar excess for 1 min and diluted to 40 μg/mL in TBS immediately before adding 3 μL to a freshly-glow discharged ultrathin, 400 mesh carbon-coated copper grid (Ted Pella, Inc.). Samples were blotted after a 1 min incubation period and stained with 1% uranyl formate for an additional minute before imaging. Micrographs were recorded on a Thermo Fisher Talos Arctica transmission electron microscope operating at 200 keV using a K3 direct electron detector (Gatan, Inc) and SerialEM automated image acquisition software38 (link). Images were acquired at a nominal magnification of 28,000x (1.44 Å/pixel size) and a −1.5 to −2.0 μm defocus range. Images were processed in cryoSPARC v2.14, and reference-free particle picking was completed using a gaussian blob picker39 (link). Reference-free 2D class averages and ab initio volumes were generated in cryoSPARC, and subsequently 3D-classified to identify classes of S-Fab complexes, that were then homogenously refined. Figures were prepared using UCSF Chimera40 (link).
5
Cryo-EM Visualization of SARS-CoV-2 S-Fab Complexes
6
Characterization of nanogel particle size and charge
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The npGG size distribution and surface charge were analyzed by dynamic light scattering after preparing a 1 mg.mL−1 suspension of rehydrated GG particles in deionized water (Zetasizer Nano ZS, Malvern Instruments, UK). Acquisition was performed with the detector positioned at a scattering angle of 173°.
Particle size and morphology were analyzed with a High-Resolution Field Emission Scanning Electron Microscope with Focused Ion Beam (FIB – SEM) (AURIGA COMPACT, ZEISS, Germany). For this effect, the samples were diluted at 0.5 mg.mL−1 in ultrapure water followed by a further dilution of 1:20 and were dispersed onto the surface of a 400-mesh carbon-coated copper grid (Ted Pella, USA) for further observation.
Particle size and morphology were analyzed with a High-Resolution Field Emission Scanning Electron Microscope with Focused Ion Beam (FIB – SEM) (AURIGA COMPACT, ZEISS, Germany). For this effect, the samples were diluted at 0.5 mg.mL−1 in ultrapure water followed by a further dilution of 1:20 and were dispersed onto the surface of a 400-mesh carbon-coated copper grid (Ted Pella, USA) for further observation.
7
Negative Staining of S1-Ferritin Nanoparticles
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Samples comprising 5 μL of purified S1-ferritin NP, S1-mi3 NP, or S1-I53-50 NP at a concentration of 0.5 mg/mL in PBS were dripped onto the surface of parafilm and adsorbed onto glow-discharged 400-mesh carbon-coated copper grids (Ted Pella) for 1 min. Excess protein solution was gently wicked away using absorbing paper. The grids were negatively stained with 2% (wt/vol) uranyl acetate for 1 min. Excess staining solution was wicked away in the same manner. Samples were imaged at 120 kV using a Talos L120C transmission electron microscope (FEI) equipped with a CETA 16M CMOS camera.
8
Characterizing Colloidal Nanoparticle Properties
9
Nanoparticle Size Analysis by TEM
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Morphological and size distribution of the NPs and nanocomplexes were determined by TEM. NPs were sonicated before being added to individual 400-mesh carbon-coated copper grids (Ted Pella Inc. Redding, CA, USA) and allowed to dry at room temperature for 30 min. The grids were then loaded onto the sample holder, and sample images were viewed in a JOEL JEM-1010 (Jeol, Tokyo, Japan) transmission electron microscope. Images were captured using iTEM Soft Imaging Systems (SIS) Megaview III fitted with a side-mounted digital camera (3-megapixels) and analyzed using analySIS LS Research v2.6 (Olympus Soft Imaging Solutions GmbH, Münster, Germany, 2004) to calculate the average particle diameter for each NP.
10
Transmission Electron Microscopy of Heated Samples
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Transmission electron microscopy (TEM) was carried out at the Cell Imaging Center at the University of Alberta. Samples for TEM were prepared as they were for temperature trend DLS measurements. The samples were heated in a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) under conditions identical to those during the temperature trend. 400 mesh carbon-coated copper grids (Ted Pella, Redding, CA, USA) were subjected to glow discharge in a Pelco easiGlow™ system (Ted Pella, Redding, CA, USA) at 0.4 mBar, 15 mAmp and positive polarity for 45 s. 10 µL samples were immediately placed on charged grids for 3 min incubations on an appropriately warmed heatblock (VWR International, Radnor, Pennsylvania, USA), before two rounds of washing in pre-warmed ultrapure MilliQ water (EMD Millipore, Etobicoke, Ontario, Canada) on the same heated surface. Unstained samples were then directly taken for TEM imaging on a Hitachi H-7650 TEM using a 60 kV accelerating voltage. Images were acquired using a 16 megapixel EMCCD camera (Advanced Microscopy Techniques) and processed using the AMT Image Capture Engine software version 602.576.
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