Sample preparation details in Supplementary. Images were acquired with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands) at an accelerating voltage of 80 kV [30 (link)].
Cm 100 transmission em
Manufactured by Philips
Sourced in Netherlands
The Philips CM 100 Transmission EM is a compact and versatile electron microscope designed for transmission electron microscopy applications. It features high-resolution imaging capabilities and is suitable for a range of sample types and research areas.
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8 protocols using cm 100 transmission em
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Transmission Electron Microscope Imaging
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Ultrastructural Analysis of MRSA Interaction
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To elucidate the interaction between JBC 1847 and MRSA USA300, transmission electron microscopy (TEM) was carried out. Bacterial cultures were inoculated in 10 ml MHB and cultivated at 37°C for 5 h, with shaking, until mid-exponential growth phase (OD550 5.5). Once mid-exponential phase was reached, the cells were treated according to the sub- and supra-MIC (0.5 × MIC, MIC, 2 × MIC, and 4 × MIC) of JBC1847 at 37°C for 1 h. An untreated control was also prepared. The five samples were placed on ice until being prepared for TEM. This included a fixation step in 2% glutaraldehyde in 0.15 M sodium phosphate buffer (pH 7.2). Subsequently, the fixed cells were washed three times and postfixed with 0.2% OsO4 (osmium tetroxide) in H2O/0.15 M sodium phosphate buffer (pH 7.2) for 1 h. The samples were then dehydrated with graded acetone, and then embedded in epoxy resin. Ultrathin sections of the samples were prepared using Formvar-coated copper grids, which were stained using 3% uranyl acetate. The samples were placed in a Philips CM 100 Transmission EM (Philips, Eindhoven, Netherlands) and exposed to 120 keV electron energy.
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Transmission Electron Microscopy Sample Preparation
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Samples were fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). Following isolation of suitable specimen blocks, the samples were rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2) and subsequently post-fixed in 1% w/v OsO4 in 0.12 M sodium phosphate buffer (pH 7.2) for 2-hour. The specimens were dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in Epon according to standard procedures. Sections, approximately 60 nm thick, were cut with a Ultracut 7 ultramicrotome (Leica, Vienna, Austria) and collected on copper grids with Formvar supporting membranes (Ted Pella, 01700-F) stained with uranyl acetate (Agar scientific, AGR1260A) and lead citrate (Agar scientific, AGR1210), and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, the Netherlands), operated at an accelerating voltage of 80 kV and equipped with an OSIS Veleta digital slow scan 2k x 2k CCD camera. Digital images were recorded with the ITEM software package. Electron microscopy was performed by Klaus Qvortrup, Professor, MD, PhD, University of Copenhagen Faculty of Health and Medical Sciences, CFIM, Copenhagen, Danmark.
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Transmission Electron Microscopy Sample Prep
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Pellets of bacteria were fixed with 2% (vol/vol) glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently postfixed in 1% (wt/vol) OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 hours. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, approximately 60-nm thick, were cut with a Ultracut 7 (Leica, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k × 2k CCD camera and the ITEM software package.
5
Transmission Electron Microscopy Sample Prep
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Pellets of bacteria were fixed with 2% (vol/vol) glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently postfixed in 1% (wt/vol) OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 hours. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, approximately 60-nm thick, were cut with a Ultracut 7 (Leica, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k × 2k CCD camera and the ITEM software package.
6
Ultrastructural Analysis of Cell Fractions
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Pellets with Hela cells, liver and kidney homogenate constituting the starting materials and pellets formed by centrifugation after each step in the fractionation protocol were fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were then embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently post-fixed in 1% w/v OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M cacodylate buffer (pH 7.2) for 2 hrs. The specimens were then dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in epon (812 Resin kit, TAAB Laboratories Equipment Ltd, Aldermaston, UK) according to standard procedures.
Sections, approximately 60 nm thick, were cut with an Ultracut 7 (Leica, Vienna, Austria) and collected on formvar coated copper grids (Electron Microscopy Sciences, Hatfield, PA), stained with 0.5 % w/v uranyl acetate and 3 % w/v lead citrate. Subsequently, they were examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera (Olympus, Münster, Germany) and the iTEM software package.
Sections, approximately 60 nm thick, were cut with an Ultracut 7 (Leica, Vienna, Austria) and collected on formvar coated copper grids (Electron Microscopy Sciences, Hatfield, PA), stained with 0.5 % w/v uranyl acetate and 3 % w/v lead citrate. Subsequently, they were examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera (Olympus, Münster, Germany) and the iTEM software package.
7
Transmission Electron Microscopy of hCMEC/D3 Cells
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All transmission electron microscopy was done at the Core Facility for Integrated Microscopy at the University of Copenhagen. hCMEC/D3 cells were grown to confluence on Thermanox coverslips and then co-cultured (4, 8, 12, or 24 h) with HB3VAR03-IEs, IT4VAR13-IEs, or noninfected erythrocytes at 37°C. Non-bound cells were washed off, and the remaining cells were fixed with 2% vol/vol glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). Following isolation of suitable specimen blocks, the samples were rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2) and subsequently post-fixed in 1% wt/vol OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 h. The specimens were dehydrated in graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, ∼60 nm thick, were cut with an Ultracut 7 (Leica) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k × 2k CCD camera and the ITEM software package.
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Electron Microscopy Cell Culture Protocol
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Cells were grown at confluence on Thermanox TM coverslips and fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). Samples were rinsed three times in 0.15 M Phosphate buffer (pH 7.2) and subsequently post-fixed in 1% w/v OsO4 in 0.12 M sodium Phosphate buffer (pH 7.2) for 2 h. The specimens were dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in Epon according to standard procedures. Following polymerization, the Thermanox TM coverslip was removed. 60 nm thick sections were cut with a Leica UC7 microtome (Leica Microsystems, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera and the ITEM software package.
Proliferation assay 1x10 4 cells were seeded per well in 24-well plates for 24, 48, 72 and 96 h in medium without antibiotics. Cells were transfected with Lipofectamine-RNAiMAX (13778100, Thermo Fisher Scientific) and re-transfected every 72 h. At the indicated time points, the cells were trypsinized, stained with 1:1 Trypan Blue to discriminate dead cells and counted in a Neubauer chamber.
Proliferation assay 1x10 4 cells were seeded per well in 24-well plates for 24, 48, 72 and 96 h in medium without antibiotics. Cells were transfected with Lipofectamine-RNAiMAX (13778100, Thermo Fisher Scientific) and re-transfected every 72 h. At the indicated time points, the cells were trypsinized, stained with 1:1 Trypan Blue to discriminate dead cells and counted in a Neubauer chamber.
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