Ruvimbo spent two weeks in UCL’s Research Department of
We wanted to see the effect of antimycin-A on the production of mitochondria derived vesicles (MDVs), a way to get rid of faulty mitochondia that contain ROS which is toxic to the cell, to compare to data provided by a published a paper about it. MDVs are vesicles have budded off the mitochondria that go to the lysosome to be destroyed.
In the experiment we had a control Cos cells (fibroblast-like cell line derived from monkey kidney tissue) in liquid with all needed nutrients, and Cos cells that were in antimycin-A and the nutrition liquid for 2 hours. The cells were on top of microscope cover-slips.
After the time the cells were washed to remove the liquid and, in the other case, the antimycin-A. Then paraformaldehyde was added for 10 minutes to the cells to fix the cells to the cover slip. Then the cells were washed again then, a block was added for 40 minutes to stop the other proteins from interfering with the antibodies.
Then water was added to an ice cube tray. We used tweezers to remove the cover slips from the dish they were in, and dunked them individually in each ice cube tray slot to wash off the excess block.
Then on parafilm we separated it into 6 sections: 3 sections were of the control where there would be the primary antibodies of rabbit Tom 20 (a mitochondrial protein) and mouse miro 1; rabbit miro 1 and finally mouse Tom 20 and rabbit miro 1. The same procedure is done for the antimycin-A. Then two drops of each primary antibody was attached to the parafilm and then the cover slips were put on top of each, dropped cell side down so they would float and we left them there for an hour. Then they were washed again with the water in the ice cube tray. After the secondary antibodies were added in the same way as the primary antibodies and dropped cell side down (so they would attach to the primary antibodies) they have a fluorescent protein attached, so they can show us what we are looking for (Tom20). They were left for 40 minutes, then washed with the water in the ice cube tray.
Then we put the cover slips on glass slides: one for control, the other for antimycin-a, and were stuck (cells facing upwards) with a mounting solution and it was left to dry under a box, so that the fluorescence would not be affected by the light. Then clear nail vanish was added to the edge of each cover slip to ensure the cover slip does not move around on the glass slide when they are viewed under the microscope.