Difference between revisions of "NJU-China-notebook.html"
Line 224: | Line 224: | ||
<div id="methods"> | <div id="methods"> | ||
<img src="https://static.igem.org/mediawiki/2015/c/c8/NJU-China_Notebook_method1.jpg" style="width:800px"> | <img src="https://static.igem.org/mediawiki/2015/c/c8/NJU-China_Notebook_method1.jpg" style="width:800px"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/1/16/%E5%AE%9E%E9%AA%8C3.jpg" style="width:400px;float:right"> | + | <img src="https://static.igem.org/mediawiki/2015/1/16/%E5%AE%9E%E9%AA%8C3.jpg" style="width:400px;float:right"> </br></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> | 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> | ||
− | <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"> </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> | 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 confocal microscopy analysis, exosomes (100 μg) loaded with fluorescence-labelled siRNA were incubated with the Neuro2A cells (10<sup>6</sup> cells). After 6 hours, the cells were washed, fixed and observed under a confocal microscope (FV 1000; Olympus, Tokyo). Pictures were taken under the following conditions: objective lens: PLAPON 60 × O; NA: 1.42; scan mode: XY; excitation wavelength: 405 nm for Hoechst 33342 and 555 nm for Alexa Fluor 555; and image size: 1024 × 1024 pixels. </br> </br> | For confocal microscopy analysis, exosomes (100 μg) loaded with fluorescence-labelled siRNA were incubated with the Neuro2A cells (10<sup>6</sup> cells). After 6 hours, the cells were washed, fixed and observed under a confocal microscope (FV 1000; Olympus, Tokyo). Pictures were taken under the following conditions: objective lens: PLAPON 60 × O; NA: 1.42; scan mode: XY; excitation wavelength: 405 nm for Hoechst 33342 and 555 nm for Alexa Fluor 555; and image size: 1024 × 1024 pixels. </br> </br> | ||
Line 245: | Line 245: | ||
<img src="https://static.igem.org/mediawiki/2015/a/af/NJU-China-ForceSwimming-title.png" style="width:900px"></br></br> | <img src="https://static.igem.org/mediawiki/2015/a/af/NJU-China-ForceSwimming-title.png" style="width:900px"></br></br> | ||
C57BL/6J Mice (n=3 for each group) were randomly divided into control group and test group. Mice in the control group received saline injection and mice in the test group received exosome injection (200 μg RVG exosomes loaded with MOR siRNA). After two days, mice were subjected to two trials during which they were plunged individually and forced to swim in a tank (height 30 cm, length 50 cm, width 40 cm) filled with 15 cm of water maintained at 25 °C. The first training trial lasted 15 min and was performed one day before testing. Then, after 24 h, a second trial was performed and lasted for 10 min. The time that the test animal spent in the second trial without making any movements beyond those required to keep its head above water was measured and further analyzed. Both of two trials were videoed. | C57BL/6J Mice (n=3 for each group) were randomly divided into control group and test group. Mice in the control group received saline injection and mice in the test group received exosome injection (200 μg RVG exosomes loaded with MOR siRNA). After two days, mice were subjected to two trials during which they were plunged individually and forced to swim in a tank (height 30 cm, length 50 cm, width 40 cm) filled with 15 cm of water maintained at 25 °C. The first training trial lasted 15 min and was performed one day before testing. Then, after 24 h, a second trial was performed and lasted for 10 min. The time that the test animal spent in the second trial without making any movements beyond those required to keep its head above water was measured and further analyzed. Both of two trials were videoed. | ||
− | <img src="https://static.igem.org/mediawiki/2015/2/21/NJU-China-methods2.png",style="width: | + | <img src="https://static.igem.org/mediawiki/2015/2/21/NJU-China-methods2.png",style="width:350px;float:left"> </br></br> |
</div> | </div> | ||
− | |||
<h3>Protocol</h3> | <h3>Protocol</h3> | ||
<div id="protocal"> | <div id="protocal"> |
Revision as of 18:31, 18 September 2015
|
Notebook
|