sEVs were isolated from CSF by differential centrifugation, according to the previous publications [35 (link)]. After removing cells and other debris by centrifugation at 2000 × g for 30 min, the supernatant was centrifuged at 12,000 × g for 45 min to remove shedding vesicles and the other vesicles with bigger sizes. The supernatant was centrifuged at 110,000 × g for 70 min and resuspended in 10 mL PBS. Finally, the suspension was recentrifuged at 110,000 × g for 70 min (all steps were performed at 4 °C); sEV were collected and resuspended in 50 μL PBS for further trans-omics RNA sequencing. The transmission electron microscopy assay of CSF sEV pellet was examined and photographed with an FEI Tecnai spirit TEM T12(FEI Tecnai Spirit 120 kv), according to the previous publications [35 (link)].
Tecnai spirit tem t12
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai Spirit TEM T12 is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of a wide range of samples. It features a 120 kV accelerating voltage and provides capabilities for both bright-field and dark-field imaging modes.
Automatically generated - may contain errors
9 protocols using tecnai spirit tem t12
1
Isolation and Characterization of CSF sEVs
2
Exosome ENTPD2 Localization by TEM
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Exosomes were washed twice with PBS, fixed with paraformaldehyde, and placed on copper grids. The grids were blocked with bovine serum albumin for 30 min, incubated with a rabbit polyclonal anti-ENTPD2 antibody for 1 h and labelled with a secondary antibody (anti-rabbit antibody conjugated to 10 nm gold grains) for 1.5 h. The grids were visualized via electron microscopy (FEI Tecnai Spirit TEM T12).
3
Exosome Isolation and Characterization from Fracture Patients
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Blood was collected in ethylenediaminetetraacetic acid-containing
tubes from male volunteers aged 20–60 years old who were diagnosed
with fracture non-healing (n = 3) or healing (n = 3) at Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology. This study was approved by the
Ethics Committee of Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology. The blood samples were centrifuged
at 3000 rpm for 15 min at room temperature (RT) and transferred to
2 mL lyophilization tubes. The isolated plasma was kept at −80
°C temporarily. Exosome purification, identification, and quantification
were performed as previously described.47 (link) Briefly, rapidly melted plasma was resuspended in PBS after several
cycles of ultracentrifugation, washing, and filtration. Exosomes were
stained with 1% phosphotungstic acid and imaged with TEM (FEI Tecnai
Spirit TEM T12). Information on particle size and concentration of
the exosomes was detected with a NanoFCM instrument (Flow Bio Flow
NanoAnalyzer). Western blotting was performed to assess the expression
of exosomal surface markers. Individual exosomes were used in different
experiments.
tubes from male volunteers aged 20–60 years old who were diagnosed
with fracture non-healing (n = 3) or healing (n = 3) at Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology. This study was approved by the
Ethics Committee of Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology. The blood samples were centrifuged
at 3000 rpm for 15 min at room temperature (RT) and transferred to
2 mL lyophilization tubes. The isolated plasma was kept at −80
°C temporarily. Exosome purification, identification, and quantification
were performed as previously described.47 (link) Briefly, rapidly melted plasma was resuspended in PBS after several
cycles of ultracentrifugation, washing, and filtration. Exosomes were
stained with 1% phosphotungstic acid and imaged with TEM (FEI Tecnai
Spirit TEM T12). Information on particle size and concentration of
the exosomes was detected with a NanoFCM instrument (Flow Bio Flow
NanoAnalyzer). Western blotting was performed to assess the expression
of exosomal surface markers. Individual exosomes were used in different
experiments.
4
Isolation and Characterization of Smad7-Loaded Extracellular Vesicles
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According to previous methods [10] , the serum starvation-induced BMSCs were transfected with 1 µg PEI25k/ pCMV5-Smad7 vector or empty PEI25k/pCMV5 vector (Addgene, Cambridge, MA, USA) for 24 h. The transfected BMSCs were cultured for 48 h. at 10,000 × g for 30 min at 4℃. Phosphate buffer saline (PBS) was used to suspend the particles, and the particles were centrifuged (100,000 × g, 120 min) twice at 4℃. The MVs were suspended in PBS and stored at -20℃. The structure of MVs was observed by a transmission electron microscopy (TEM, FEI Tecnai Spirit TEM T12, USA) and the diameter of MVs was analyzed by a qNano particle analyzer (#ZEN3690, Malvern instruments limited, UK). The specific MVs markers, CD9 and CD63, were measured by western blotting. The delivery of Smad7 in MVs was assessed by western blotting.
5
Isolation and Characterization of Plasma Exosomes
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Exosomes were isolated using ExoEasy Maxi Kit (Qiagen, 76064, USA). Briefly, 2 ml plasma samples filtered using a 0.8 μm filter syringe (Millipore, SLAA033SB, USA) are mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with buffer XWP, eluted with 400μl Buffer XE (an aqueous buffer containing primarily inorganic salts). The exosome samples were stored at −20°C until further detection. Exosomes were observed by transmission electron microscopy (TEM) (FEI, Tecnai Spirit TEM T12). The size and concentration of the exosomes were assessed using Flow NanoAnalyzer (NanoFCM, Xiamen, China). Exosomal proteins CD81 (BD, 551108, USA) and CD9 (BD, 555317, USA) expression were measured using Nano-Flow analysis (NanoFCM, Xiamen, China).
6
TEM Imaging of Ultrathin Resin Sections
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Resin‐embedded ultrathin 70 nm sections on copper grids were imaged on a FEI Tecnai T12 Spirit TEM. Images were acquired in a CCD camera (Eagle).
7
Hsp27 Protein Structural Analysis
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Samples of Hsp27 (1 µM, in SEC buffer) were negatively stained with uranyl formate (pH ~6.0) on thin-carbon layered 400-mesh copper grids (Ted Pella) that were glow discharged before sample was applied. Samples were imaged using a Tecnai T12 Spirit TEM (FEI) operated at 120 keV. Micrograph images were acquired with ∼1.5-µm defocus on a 4k × 4k CCD camera (Gatan) at a magnification of 67,000× with a pixel size of 1.73 Å.
8
Oxidative Effects on LACTB Protein
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An aliquot of purified LACTB was diluted to 0.15 mg ml-1. Aliquots of 10 μl of the protein sample were incubated with 5 μl of varying concentrations of H2O2 for 30 min. The final concentrations were 0.01% H2O2 (v/v), 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, and 1.0%. The final concentration of protein was 0.1 mg ml-1. The samples were analyzed by SDS PAGE using a 4% to 15% precast gel (BioRad) at 100 V. The 0.1%, 0.5%, and 1.0% H2O2 (v/v) samples were stained on glow-discharged carbon-coated copper grids with UF as described above and visualized with a Tecnai T12 Spirit TEM (FEI) operated at a voltage of 100 kV. The micrographs were collected at a nominal magnification of 120,000× at the specimen level with a calibrated pixel size of 5.28 Å per pixel.
9
High-Pressure Freezing and Ultrastructural Analysis
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High-pressure freezing (HPF) and freeze substitution sample preparation were performed at the Microscopy and Histology Facility at the University of Aberdeen, as described previously (60 (link)). Briefly, cells were washed 3 times in water, and then HPF was performed in a Leica EM PACT 2 (Leica Microsystems, Milton Keynes, UK). Freeze substitution was performed in a Leica AFS 2 in acetone-1% OsO4. Samples were transferred to epoxy resin, and ultrathin sections were cut, stained with uranyl acetate and lead citrate, and imaged with an FEI Tecnai T12 Spirit TEM with Gatan camera. Cell walls were measured via FIJI with 5 measurements averaged per cell (n > 20 cells per condition).
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