Isolated cilia in solution were applied on glow-discharged TEM grids with holey carbon support film (Quantifoil Micro Tools, R 3.5/1). Colloidal gold particles (10 nm) were added to the sample to be used as fiducial markers during tomogram reconstruction. The samples were blotted from the back with Whatman filter paper, rapidly frozen in liquid ethane at −182°C using a Leica EM Grid Plunger, and stored in liquid nitrogen until image acquisition.
R3.5 1
Manufactured by Quantifoil
Sourced in Germany
The Quantifoil R3.5/1 is a carbon support film with a regular array of circular holes. It is designed for use in electron microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (2 mentions)
Vitrobot, by Thermo Fisher Scientific (2 mentions)
Cm 120 cryo electron microscope, by Philips (2 mentions)
Titan krios, by Thermo Fisher Scientific (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Ultrascan 4000 ccd camera, by Ametek (1 mentions)
Em gp automatic plunge freezer, by Leica (1 mentions)
Whatman paper, by Cytiva (1 mentions)
Probe sonicator, by Emerson (1 mentions)
9 protocols using r3.5 1
1
Cryo-TEM Imaging of Isolated Cilia
2
Centriole Isolation and Cryo-Electron Tomography
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Centriole isolation and tomogram acquisition were performed as previously described in (Le Guennec et al, 2020 ). Briefly, the N. gruberi NEG strain was differentiated into flagellates (Fulton, 1977 ), and centrioles were isolated using a sucrose gradient. Isolated centrioles were then deposited onto 200‐mesh copper EM grids coated with holey carbon (R3.5/1, Quantifoil Micro Tools) and plunge‐frozen in a liquid ethane/propane mixture. Tilt series were recorded using SerialEM (Mastronarde, 2005 ) on a 300 kV FEI Titan Krios transmission electron microscope, equipped with a direct detector camera (K2 Summit, Gatan) and a post‐column energy filter (Quantum, Gatan). Tilt series were bidirectional (2° steps, separated at −0° or −20°), and individual images were recorded in movie mode at 10 frames/s, with an object pixel size of 4.21 Å and a defocus of −5 to −8 μm.
3
Cryo-ET of C. trachomatis Infection
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A total of 2 × 106 HeLa or U2OS cells were seeded into well plates and cultured overnight. Cells were then infected using frozen aliquots of C. trachomatis LGV2 (moi 5) by centrifugation-assisted inoculation. The following day, non-infected cells (HeLa or U2OS) were seeded on 200 mesh gold EM grids in a separate well plate (R3.5/1; Quantifoil Micro Tools, Jena, Germany) at a density of 1 or 2 cells per grid square. At ∼ 48 hpi when infected cells begin to release new EB progeny, the EM grids were introduced to the tissue culture dish and incubated (15 min to 1 h at 37°C) so that the cells could be infected directly with the released EBs. Grids were then removed and rinsed in Hank's buffered salt solution (HBSS). Four microlitres of BSA-coated 10 nm colloidal gold (Sigma) was added to the grid before it was plunge-frozen into liquid ethane (Vitrobot Mark III, FEI). Frozen grids were stored in liquid nitrogen until they were loaded into the electron microscope for cryo-electron tomography (see also Fig. S1 ).
4
Manual Plunge-Freezing of SaR2lox Crystals
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A cryo-EM sample of SaR2lox was prepared by freezing the crystals in a thin layer of vitrified ice. A thin and uniform vitrified ice layer is crucial for obtaining MicroED of high signal-to-noise ratio. Meanwhile, the ice layer has to protect the crystals from being dehydrated under vacuum inside a TEM. The 4-μl hanging drop was deposited onto a QUANTIFOIL R 3.5/1 (300 mesh) Cu holy carbon TEM grid. The excessive liquid was removed by manual back-side blotting. The grid was then rapidly plunge-frozen in liquid ethane. We note that the automated blotting and vitrification routine using a FEI Vitrobot Mark IV was not efficient in removing the viscous mother liquid while leaving a sufficient number of crystals on the TEM grid.
5
Cryo-EM Sample Preparation Protocol
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A 3 mL aliquot of sample solution was pipetted on to plasma-treated (Gatan Solarus Model 950 Advanced Plasma System, p = 70 mTorr, H2 flow 6.4 sccm, O2 flow 27.5 sccm, orward RF target 50 W, time 30 s) carbon copper grids (Quantifoil R 3.5/1) in the environmental chamber of a fully automated vitrification device for plunge freezing (FEI Vibrot), having relative air humidity of 100% and a temperature of 22 °C. The excess solution was removed by blotting with filter paper for 2 s followed by 1 s draining and plunging of the samples into 1:1 mixture of liquid ethane and liquid propane, which was cooled to −170 °C. Vitrified samples were cryo-transferred into a Jeol JEM3200FSC cryo-TEM operating at −194 °C. The temperature of the samples was −187 °C during image acquisition. The microscope was operated in the bright field mode, using a 300 kV acceleration voltage; the in-column energy filter was set to 0–20 eV energy-loss range (zero-loss imaging). Micrographs were recorded with a Gatan Ultrascan 4000 CCD camera.
6
Negative Staining and Cryo-EM Sample Preparation
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For negative stain analysis the cross-linked sample was diluted in size-exclusion buffer to around 0.2 mg/mL. A 3.5 μL aliquot of the sample was loaded onto a freshly glow discharged thin carbon-coated grid (Quantifoil R3.5/1 with a 2 nm carbon coating), incubated for 45 s at room temperature and washed with six drops of size-exclusion buffer. The grid was then stained with a drop of 2% uranyl-acetate for 15 s and dried on a filter paper.
For cryo-EM grids, the cross-linked complex was diluted to around 0.4 mg/ml. Four microliters of the sample were loaded on a previously glow discharged holey carbon (Quantifoil R2/1) grid. After 1 s of blotting time, grids were plunge frozen in liquid ethane by using a Leica EM GP-Automatic Plunge Freezer. The temperature in the chamber was kept at +15°C and the humidity at 95%.
For cryo-EM grids, the cross-linked complex was diluted to around 0.4 mg/ml. Four microliters of the sample were loaded on a previously glow discharged holey carbon (Quantifoil R2/1) grid. After 1 s of blotting time, grids were plunge frozen in liquid ethane by using a Leica EM GP-Automatic Plunge Freezer. The temperature in the chamber was kept at +15°C and the humidity at 95%.
7
Cryo-EM Sample Preparation of Vaccinia Virus
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HeLa cells were maintained in complete MEM (supplemented with 10% fetal bovine serum, 100 ug/ml streptomycin, and 100 U/ml penicillin) at 37 °C with 5% CO2. Cells were washed with PBS, treated with trypsin, seeded on glow discharged (40 s at 45 mA) Quantifoil R3.5/1 gold grids of 200 mesh and placed in wells of 6-well plates with complete MEM. After overnight growth, the cells were infected with A36-YdF ΔF11 or A36-YdF ΔNPF1-3 ΔF11 vaccinia strains in serum-free MEM at a multiplicity of infection of 2. After 1 h, the medium was replaced with complete MEM. At 8 h post infection, grids were washed once with PBS and excess PBS was removed with a Whatman paper before blotting and vitrification using the Vitrobot Mark IV System, which was set to 95% relative humidity at 22°C. Colloidal gold particles (10-nm diameter) were pipetted onto grids before blotting on both sides of the grid, which was performed for 14 s with a relative force of −10.
8
Cryo-TEM Analysis of Surfactant-Coated Nanogels
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Cryogenic transmission electron microscopy (cryo-TEM) is a valuable tool for the structural investigation of nanosized particles [38] . Both NGs and Curosurf ® vesicles were diluted in HEPES buffer (final concentration of respectively 5 and 10 mg mL -1 ). The Curosurf ® vesicles were formed by sonication of the crude surfactant dispersion using a probe sonicator (amplitude 10 %; Branson). Coated nanogels were formed according to the method described above. The ratio of surfactant to NGs equaled 7.5 mg Curosurf ® / mg NGs. 2.5 µL of the different samples was placed on a glow discharged holey carbon coated grid (Quantifoil ® R 3.5/1, Quantifoil Micro Tools GmbH, Grosslöbichau, Germany). The excess of liquid was blotted with a filter paper, reducing the droplet to a thin film. The samples were vitrified in liquid ethane, using a vitrobot (FEI, Eindhoven, The Netherlands) and stored in liquid nitrogen until observation. The grids were transferred to a cryo specimen holder type 626 (Gatan GmbH, München, Germany) and observed in a CM 120 cryo-electron microscope (Philips, Eindhoven, The Netherlands) operating at 120 keV. Images were recorded on a slow-scan CCD camera (Gatan GmbH) under low-dose conditions.
9
Cryo-EM Characterization of Coated Nanoparticles
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Coated NGs were formed according to the method described above. The ratio of surfactant to NGs equaled 7.5 mg Curosurf ® per mg NGs. 2.5 µL of the different samples was placed on a glow discharged holey carbon coated grid (Quantifoil ® R 3.5/1, Quantifoil Micro Tools GmbH, Grosslöbichau, Germany). The excess of liquid was blotted with a filter paper, reducing the droplet to a thin film. The samples were vitrified in liquid ethane, using a vitrobot (FEI, Eindhoven, The Netherlands) and stored in liquid nitrogen until observation. The grids were transferred to a cryo specimen holder type 626 (Gatan GmbH, München, Germany) and observed in a CM 120 cryo-electron microscope (Philips, Eindhoven, The Netherlands) operating at 120 keV. Images were recorded on a slow-scan CCD camera (Gatan GmbH) under low-dose conditions.
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