HMEECs were cultured on glass cover slips and were infected with bacteria for varying time periods. After incubation, the cells were washed 5 times with warm phosphate buffered saline (PBS, pH 7.4, Cellgro, Manassas, VA)) buffer to remove unbound bacteria and were then processed for SEM. Samples were fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS buffer followed by three changes of PBS buffer for 10 min each. The samples were then post–fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in PBS buffer for 45 min and rinsed in three changes of PBS buffer for 10 min each. The samples were dehydrated in a graded series of ethanol, dried in hexamethyldisilazane (HMDS) (Electron Microscopy Sciences, Hatfield, PA) and mounted on carbon adhesive tabs fixed to metal stubs. The samples were coated with palladium in a plasma sputter coater and viewed in a scanning electron microscope (FEI, ESEM-FEG XL-30).
Hexamethyldisilazane hmds
Manufactured by Electron Microscopy Sciences
Sourced in United States
Hexamethyldisilazane (HMDS) is a silicon-containing compound used in various laboratory applications. It is a colorless liquid with a characteristic odor. HMDS acts as a drying and dehydration agent, facilitating the preparation of samples for electron microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Electron Microscopy Sciences (3 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (2 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Esem xl 30 feg, by Thermo Fisher Scientific (1 mentions)
Colloidal silver, by Electron Microscopy Sciences (1 mentions)
Nitrate nitrite colorimetric assay kit, by Cayman Chemical (1 mentions)
Vasculife basal medium, by Lifeline Cell Technology (1 mentions)
Cyquant ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Vasculife vegf lifefactors kit, by Lifeline Cell Technology (1 mentions)
3 protocols using hexamethyldisilazane hmds
1
Scanning Electron Microscopy of Bacteria-Infected HMEECs
2
Biofilm Imaging by Scanning Electron Microscopy
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For all single species biofilms, as well as any 2-genera biofilms wherein each pathogen had a distinct cellular morphology, we determined biofilm architecture and spatial relationships by SEM, as we could easily identify each pathogen visually. As such, single species and 2-genera biofilms were allowed to form as described above in 8-well chambered coverglass culture vessel into which a sterile 12 mm glass coverslip had been inserted prior to incubation. Biofilms were allowed to form for 16 h prior to overnight fixation at 4°C in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, Pa) in 0.1 M phosphate buffer pH 7.4. Sixteen hours was chosen as the incubation period for all SEM imaging to allow ready discrimination of each bacterium and their relationship to each other before increased biofilm density obscured this detail. Samples were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2 (Electron Microscopy Sciences), dehydrated slowly through a series of graded ethanol solutions, then dried overnight using hexamethyldisilazane (HMDS) (Electron Microscopy Sciences). Coverslips were mounted onto 15 mm aluminum stubs, sealed with colloidal silver (Electron Microscopy Sciences) and air dried. Dried stubs were sputter coated with 2 nm gold/palladium and imaged using a Hitachi S-4800 SEM/TEM.
3
Biocompatible PCL Scaffold Characterization
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All chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) if not mentioned otherwise. Medical grade PCL filament was purchased from Advanced Biomedical Technology Inc. (Hsinchu City, Taiwan). The Nitrate/Nitrite Colorimetric Assay Kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). The CyQUANT™ LDH Cytotoxicity Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The VascuLife® Basal Medium and the VascuLife® VEGF LifeFactors Kit were purchased from Lifeline Cell Technology (Frederick, MD, USA). The NucSpot® 470 Nuclear Stain, 1000x in DMSO, was purchased from Biotium, Inc. (Fremont, CA, USA). The EdU-Click 594 Cell Proliferation Kit was purchased from baseclick GmbH (Munich, Germany). The lab-grade BaSO4 was purchased from Lab Alley (Spicewood, TX, USA). The acetone was purchased from Lab Chem Inc. (Zelienople, PA, USA). The hexamethyldisilazane (HMDS), as well as the glutaraldehyde, were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). The phosphate buffered saline (PBS) was purchased from Life Technologies Limited (Paisley, PA, USA). The formaldehyde was purchased from Alfa Aesar Thermo Fisher Scientific (Ward Hill, MA, USA).
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