The integrity of MS2 phages after EDA–CDots treatment was examined by TEM imaging. MS2 phage samples were treated with 50 μg mL−1 CDots under the visible light or in the dark for 2 h. The samples were loaded on copper grids and air dried, followed by a negative staining step by placing uranyl acetate on the grids for 30 s. The excess stain was wicked with filter paper and the grids were air dried. TEM imaging of MS2 samples was performed in the Microscopy Services Laboratory Core Facility at the University of North Carolina (UNC) at Chapel Hill School of Medicine, using a JEOL JEM-1230 transmission electron microscope operating at 80 kV (JEOL USA, Peabody, MA), equipped with the Gatan Orius SC1000 CCD camera and Gatan Microscopy Suite 3.0 software (Gatan, Inc., Pleasanton, CA).
Gatan microscopy suite 3
Manufactured by Ametek
Sourced in United States
The Gatan Microscopy Suite 3.0 software is a comprehensive platform for the analysis and processing of electron microscopy data. It provides a suite of tools for image acquisition, visualization, and advanced data processing, enabling users to extract valuable information from their electron microscopy samples.
Automatically generated - may contain errors
Lab products found in correlation
Jem 1230 transmission electron microscope, by JEOL (3 mentions)
Orius sc1000 ccd camera, by Ametek (2 mentions)
Formvar carbon coated 400 mesh copper grid, by Ted Pella (1 mentions)
Jem 1230, by JEOL (1 mentions)
Six well tissue culture treated plates, by Corning (1 mentions)
Spurr s epoxy resin, by Polysciences (1 mentions)
Orius sc1000 ccd digital camera, by Ametek (1 mentions)
4 protocols using gatan microscopy suite 3
1
Evaluating MS2 Phage Integrity after CDots Treatment
2
Phage Preparation for Transmission Electron Microscopy
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Phages were prepared for TEM using a modification of the method described by Fortier and Moineau (35 (link)). Prior to observation, 1.5 ml of crude lysate was centrifuged for 1 h at 4°C and 24,000 × g. A fraction of the supernatant (approximately 1.4 ml) was gently removed and discarded, and 1 ml of ammonium acetate (0.1 M, pH 7.5) was added to the remaining lysate, which was then centrifuged as described above. This step was performed twice. Washed phage samples were visualized by negative-stain transmission electron microscopy. A glow-discharged Formvar/carbon-coated 400-mesh copper grid (Ted Pella, Inc., Redding, CA) was floated on a 25-μl droplet of the sample suspension for 5 min and transferred quickly to 2 drops of deionized water, followed by transfer to a droplet of 2% aqueous uranyl acetate stain for 30 s. The grid was blotted with filter paper and air dried. Samples were observed using a JEOL JEM-1230 transmission electron microscope operating at 80 kV (JEOL USA, Peabody, MA), and images were taken using a Gatan Orius SC1000 charge-coupled-device camera with Gatan Microscopy (suite 3.0) software (Gatan, Inc., Pleasanton, CA).
3
Visualizing Mycobacterial Infection and Phage Treatment
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THP-1 and A549 cells were seeded in six-well tissue culture-treated plates (Corning) at 1 × 106 and 2.5 × 104 cell/mL, respectively. Mammalian cells were first infected with M. abscessus at an MOI of 10 for 3 h. Following three washes to remove extracellular bacteria, fresh media containing phage at an MOI of 104 and 50 µg/mL amikacin were added to the M. abscessus-infected monolayer. After 24 or 48 h, the infected monolayers were washed three times with PBS, followed by a 3-minute incubation with phage inactivation buffer (43 (link)) (PIB; 40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3.0), and three additional washes with PBS. Cells were then fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4, for 1 h at room temperature and stored at 4°C. Cells were further processed and stained with uranyl acetate, followed by Reynolds’ lead citrate (54 (link)), as described in the supplemental methods. Samples were observed with a JEOL JEM-1230 transmission electron microscope operating at 80 kV (JEOL USA, Peabody, MA), and digital images were acquired using a Gatan Orius SC1000 CCD camera and Gatan Microscopy Suite 3.0 software (Gatan, Inc., Pleasanton, CA).
4
Ultrastructural Analysis of Arabidopsis Tissues
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Arabidopsis cotyledons and hypocotyls grown ¼ MS liquid media in aluminum foil‐covered plates were illuminated with ~480 μmol m−2 s−1 light for 10 min and immersion‐fixed in 4% paraformaldehyde/3% glutaraldehyde/0.05 M sodium phosphate, pH 7.4, overnight at 4°C. After three buffer washes (w/0.05 M NaPhos), the samples were postfixed in 1% osmium tetroxide/0.05 M sodium phosphate, pH 7.4, for 1 hr followed by three washes in deionized water. Cotyledons and hypocotyls were stained en bloc with 2% aqueous uranyl acetate, dehydrated through a graded series of ethanol (30%, 50%, 75%, 90%, 100% X3) and propylene oxide, infiltrated, and embedded in Spurr's epoxy resin (Polysciences, Warrington, PA). Using a diamond knife, 1‐μm longitudinal and transverse sections were cut, mounted on slides, and stained with 1% toluidine blue and examined by light microscopy to isolate the region of interest. Ultrathin sections (70–80 nm) were cut with a diamond knife and mounted on 200 mesh copper grids followed by staining with 4% aqueous uranyl acetate for 12 min and lead citrate for 8 min. Grids were observed using a JEOL JEM‐1230 transmission electron microscope operating at 80 kV (JEOL USA, Inc., Peabody, MA), and images were acquired with a Gatan Orius SC1000 CCD Digital Camera and Gatan Microscopy Suite 3.0 software (Gatan, Inc., Pleasanton, CA).
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