Exosomes were suspended in 100 μL PBS and were fixed with 5% glutaraldehyde at incubation temperature and then maintained at 4°C until TEM analysis. According to the TEM sample preparation procedure, we placed a drop of exosome sample on a carbon-coated copper grid and immersed it in 2% phosphotungstic acid solution (pH 7.0) for 30 s. The preparations were observed with a transmission electron microscope (Tecnai G2 Spirit Bio TWIN; FEI, USA).
G2 spirit biotwin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Netherlands
The G2 Spirit BioTWIN is a compact and versatile centrifuge designed for a wide range of laboratory applications. It features a brushless motor and a robust construction, ensuring reliable and efficient operation. The centrifuge can accommodate both fixed-angle and swing-out rotors, allowing for flexible sample processing. Its user-friendly interface and intuitive controls make it easy to operate.
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Lab products found in correlation
Eagle 4k hs digital camera, by Thermo Fisher Scientific (3 mentions)
Nano zs90, by Malvern Panalytical (1 mentions)
Cary 5000, by Agilent Technologies (1 mentions)
Durcupan epoxy resin, by Merck Group (1 mentions)
Uct ultramicrotome, by Leica (1 mentions)
1200 ex 2, by JEOL (1 mentions)
Cf400 cu, by Electron Microscopy Sciences (1 mentions)
Nanosizer technology, by Malvern Panalytical (1 mentions)
Veleta camera, by Olympus (1 mentions)
18 protocols using g2 spirit biotwin
1
Exosome Morphology Characterization by TEM
2
Transmission Electron Microscopy of B. cereus
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B. cereus cells were grown and incubated with AMPs as described above for SEM sample preparation. Cell pellets were obtained from 10 mL of each control or treated cell suspension and fixed with 2.5% buffered glutaraldehyde. The cells were post-fixed with 1% osmium tetroxide for 2 h at room temperature. This was followed by dehydration in a graded ethanol series (70%, 80%) for 1 h and ‘en-bloc’ staining with 1% uranyl acetate in 95% ethanol for 1 h. The final dehydration was carried out in absolute alcohol for 30 min at 4 °C. The clearing was done with propylene oxide at room temperature. The cells were then processed for infiltration; wherein propylene oxide was replaced with liquid resin araldite CY212. Infiltration was carried out on a rotator in two steps: a mixture of propylene oxide and araldite (1:1) overnight and two changes in pure araldite for 2 h at room temperature. Cells were allowed to polymerize in an oven at 60 °C for 48 h. Finally, 40–60-nm-thick sections were collected on 300 mesh copper grids, stained with lead citrate, and were viewed under the transmission electron microscope (FEI, TECNAI G2 Spirit BioTwin, Netherlands).
3
Physicochemical Characterization of TiO2-Ce6-CpG
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The dynamic particle size and zeta potential of TiO2-Ce6-CpG were measured by dynamic light scattering (DLS) (NanoZS 90, Malvern, USA). The morphology and structure of nanosonosensitizer were observed by transmission electron microscopy (TEM) (Tecnai G2 Spirit BioTwin, FEI, USA). To test the encapsulation efficiency (EE), Ce6 and CpG in the nanosonosensitizer were respectively detected by UV-vis-near-infrared spectroscopy (UV-vis-NIR, Cary 5000, Agilent, USA) to calculate the EE as the formula: EE (%) = ((weight of loaded drug)/(weight of initially added drug)) × 100. Accordingly, the drug loading (DL) was calculated as the formula: DL (%) = ((weight of loaded drug)/(weight of nanosonosensitizer)) × 100.
4
Exosome Ultrastructural Analysis
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Exosomes were suspended in 100 μL of PBS and were fixed with 5% glutaraldehyde at incubation temperature and then maintained at 4°C until TEM analysis. According to the TEM sample preparation procedure, we placed a drop of exosome sample on a carbon-coated copper grid and immersed it in 2% phosphotungstic acid solution (pH 7.0) for 30 s. The preparations were observed with a transmission electron microscope (Tecnai G2 Spirit Bio TWIN; FEI, USA).
5
Electron Microscopy of VSMC Mitochondria
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VSMCs were digested, centrifuged, and fixed by glutaraldehyde overnight. Subsequently, the specimens were fixed by 1% osmium tetroxide for 1 h, progressively dehydrated with rising concentrations of ethanol, and finally embedded in TAAB Epon (Marivac, Canada). The ultrathin sections (60 nm) were prepared on copper grids and stained with 1% uranyl acetate and lead citrate. Images were taken under TEM (Tecnai G2 Spirit BioTWIN; FEI, USA) at an accelerating voltage of 80 kV. Mitochondrial parameters including length, width, area, and length to width ratio were measured using MetaMorph software in five randomly selected fields. The mitophagy number was counted in the whole cell.
6
Ultrastructural Analysis of Interscapular BAT
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Interscapular BAT and small tissue fragments were immersed in modified Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) for at least 4 h, postfixed in 1% osmium tetroxide in 0.15 M cacodylate buffer for 1 h, and stained en bloc with 2% uranyl acetate for 1 h. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 min and Sato's lead stain for 1 min. Grids were viewed using a JEOL 1200EX II (JEOL) transmission electron microscope and photographed using a Gatan digital camera (Gatan) or viewed using a Tecnai G2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4k HS digital camera (FEI). Mitophagosomes were quantified as described by Duan et al. (47 ).
7
Exosome Morphology Characterization by TEM
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Exosomes were suspended in 100 μL PBS and were fixed with 5% glutaraldehyde at incubation temperature and then maintained at 4°C until TEM analysis. According to the TEM sample preparation procedure, we placed a drop of exosome sample on a carbon-coated copper grid and immersed it in 2% phosphotungstic acid solution (pH 7.0) for 30 s. The preparations were observed with a transmission electron microscope (Tecnai G2 Spirit Bio TWIN; FEI, USA).
8
Transmission Electron Microscopy of HEK 293 Cells
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The fixed, resin-embedded, cross-sections from untransfected or transfected HEK 293 cells were characterized with a Tecnai G2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4k HS digital camera (FEI). The resulting transmission electron microscopy images were analyzed with ImageJ (v 2.0.0-rc-69/1.52i).
9
Negative Staining of sCCVs
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sCCVs were adsorbed for 60 s onto freshly glowed-discharged carbon-coated electron microscope grids, washed with a few drops of Milli-Q water, stained for 30 s with a few drops of 1.2% uranyl acetate, and blot dried. The samples were imaged on a Tencai G2 Spirit BioTWIN (FEI) at 23,000–49,000× magnification.
10
Ultrastructural Analysis of N. fowleri
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For ultrastructural analysis, 2 × 106N. fowleri trophozoites were incubated with 0.5% DMSO, 6.2 μM of ebselen, 1.6 μM of BAY 11-7082 and 2.3 μM of BAY 11-7085 for 24 and 48 h and then fixed with a solution of modified Karnovsky's fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate, pH 7.2). Samples were post-fixed with 1% (w/v) osmium tetroxide, dehydrated with ethanol and propylene oxide and embedded in epoxy resin. Thin sections (50–60 nm) were contrast stained with 2% uranyl acetate followed by Sato's lead stain for 1 min and thin sections were examined under a Tecnai G2 Spirit BioTWIN transmission electron microscope (TEM) equipped with an Eagle 4k HS digital camera (FEI, Hilsboro, OR).
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