Difference between revisions of "NJU-China-notebook.html"
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HEK293 cells were seeded in 225-cm<sup>2</sup> flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Lamp2b-RVG and MOR siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes loaded with MOR siRNA were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS. </br> | HEK293 cells were seeded in 225-cm<sup>2</sup> flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Lamp2b-RVG and MOR siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes loaded with MOR siRNA were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS. </br> | ||
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<img src="https://static.igem.org/mediawiki/2015/4/42/NJU-China_Notebook_method2.jpg" style="width:800px;height:200px"> | <img src="https://static.igem.org/mediawiki/2015/4/42/NJU-China_Notebook_method2.jpg" style="width:800px;height:200px"> | ||
For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm<sup>3</sup>). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV. </br></br> | For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm<sup>3</sup>). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV. </br></br> |
Revision as of 17:43, 18 September 2015
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Notebook
Protocol |