The morphology of treated and untreated MC3T3-E1 cells was also examined using TEM analysis. For that, cells from each sample were washed with PBS, fixed with 0.1 M sodium cacodylate buffer (pH, 7.2) containing 2% glutaraldehyde, and stored at 4 °C for one week. After that, the cells were fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at room temperature. Subsequently, the specimen were dehydrated in an ethanol series, embedded in polybed 812 resins (Polyscience, Warrington, PA) and polymerized for 72 h at 60 °C. Samples were sectioned into thin slices using Sorvall MT 5000 ultramicrotome (Sorwall, Norwalk, CT). The thin sections were then post-stained with saturated uranyl acetate and Reynold's lead citrate, each for 8 min before viewing with a JEOL 1200 EX TEM microscope (JEOL, Peabody, MA).
1200 ex tem microscope
Manufactured by JEOL
The JEOL 1200 EX TEM is a transmission electron microscope designed for high-resolution imaging and analysis of materials at the nanoscale. It features a LaB6 electron source and offers a maximum accelerating voltage of 120 kV. The microscope is capable of providing detailed structural and compositional information about a wide range of samples.
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2 protocols using 1200 ex tem microscope
1
Morphological Analysis of MC3T3-E1 Cells
2
Isolation and Ultrastructural Analysis of Mononuclear Cells
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Human heparinized whole blood (diluted 1:1 with phosphate buffered saline, PBS) was layered on Lymphosep (MP Biomedicals) and centrifuged for 30 min (700 g). Mononuclear cells were collected from interphase and washed in PBS and Roswell Park Memorial Institute medium (RPMI 1640) with 10% fetal calf serum (FCS). Cells were adjusted to 2.5 × 106 cells/mL in RPMI, 10% FCS, and pipetted in sixplicates in a volume of 180 µL to the microplates. MNPs in concentrations 0.12, 3, and 75 µg/cm2 were added in a volume of 20 μL. The plates were incubated at 37°C for 24 h under 5% CO2 atmosphere. Then the cells were two times washed with saline, centrifuged, saline was decanted, and the cells were fixed with 2.5% glutaraldehyde in PBS (pH 7.2) at room temperature for 60 min. Subsequently, the cells were washed with PBS and dehydrated with increasing concentration of ethanol (30, 50, 70, 90, 2 × 100%) in the solution. The cell pellet was embedded in London Resin White (Polysciences; Warrington, PA, USA) and polymerized at 60°C for 24 h. Ultrathin sections were prepared using LKB ultramicrotome and captured on EM mesh without the support membrane without staining. Ultrathin sections were evaluated using a JEOL 1200 EX TEM microscope with 100 kV accelerating voltage.
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