Seeding and Transfection for 2nd and 3rd Trials

Yesterday, I successfully seeded HeLa cells into a six-well plates for my 2nd trial. Before seeding, I ran into a small issue. Over the weekend, my HeLa cells have multiplied so much that they are now growing on top of each other. With my PI’s help, I proceeded with trypsinizing the overgrown flask, but this time I diluted the cells with double the amount of medium I was supposed to use. This technique has yielded satisfactory results, as I was able to see a nice population of cells in each of my wells under the microscope this morning. After viewing my six-well plates, I continued on with transfection. Similar to my 1st trial, I am now transfecting my cells with PMT2, MK-STYX, and F1 (active mutant of MK-STYX that has dephosphorylation capability). Although the 1st trial from last week was successful, I did notice a decrease in transfection efficiency. This might have resulted from the fact that I transfected the cells 22 hours after seeding. To increase my transfection efficiency, I decided to transfect the cells ~18 hours after seeding this time. I’m hoping that I would be able to see more cells transfected than last time so that I would be able to see more of the effects that MK-STYX exerts on the localization of the autophagy regulator TFEB (transcription factor EB). Today, I will also be seeding another six-well plates from a different flask. I have already viewed the flask this morning and there is a nice population inside the flask, so I will only need to trypsinize the cells and will not need to dilute them. At this point, I am paying much more attention to every step of my procedure, making sure to reduce any errors that might possibly contribute to not providing the maximal data.