Microscopy Tips

Last week, I went back into my first successful trial and tried to score the the slides using fluorescent microscopy. I found that most of the cells on my of my slides are now gone and that the slide could no longer be scored. I recall that when I first viewed this slide under the microscope last week, it took a few minutes for the camera to snap a picture. The reason for this is that the images that I was seeing in the camera driver is now real and was not visible with the naked eye. The time that was required to snap a picture of my slide had caused my fixed cells to become photobleached. This is extremely disappointing, because the successful trial cannot yield satisfactory data for my project. Despite this, I am now aware of a new microscopy tip. Since pictures can be taken at any time, I should score my slides as soon as possible and wait to take the pictures later. At this point, I plan on repeating this trial in order to score the cells and get a good sense of how MK-STYX is causing HeLa cells to respond to stress (serum-starve).

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.

More on Troubleshooting and 1st Successful Trial

With all my cells dying lately, I have began to feel that I might not be able to see any successful experiment during this summer. However, a last minute consultation with my PI has allowed me to alter a small portion of my protocol to yield a successful experiment. With my past experiments, I have been using DMEM with 0% FBS as the medium for my HeLa cells when I serum-starve them. After seeing that my cells keep dying after less than 18 hours of serum-starving, my PI has advised me to switch my choice of medium. So instead of using DMEM with 0% FBS, I am now using DMEM with 0.1% FBS. This gives my HeLa cells just a bit more nutrient and allows them to survive during the overnight starvation period, as well as go into autophagy mode. This worked out perfectly, as my cells survived after an overnight period of starvation and I was able to continue on with fixation. During fixation, I also made sure that my cells remained hydrated. I have been doing this by not completely aspirating away all the D-PBS during my wash steps. In the past, I have noticed that not all my cover slips were completely submerged in D-PBS when I am adding in more D-PBS after aspirating them out. This time, I also made sure that each coverslip are entirely submerged in the D-PBS at all times because my cells are facing up, so it is crucial that the coverslips are submerged. So far, these small adjustments have allowed my cells to survive and give rise to my 1st successful experiment of the summer. I was able to see the results of TFEB localization in HeLa cells upon serum-starvation and in the presence of MK-STYX as of Friday last week. I am extremely happy with the data that I’ve obtained and am looking forward to having more successful experiments this week.

Troubleshooting is a pain, but a worthwhile one…

While running my 3rd and 4th trials for my experiment on TFEB localization in the presence of the pseudophosphatase MK-STYX, I have noticed that all my cells have died and that I will need to re-run my 3rd and 4th experiments in order to get data. The fact that my experiments have failed once again was disappointing and coming to terms with the fact that I will have to spend at least a couple more weeks before I can get my data been tough, but I have come to realize that the only way to get over this hill is to sit down and troubleshoot. I went back into my notebook and looked over every step that I performed and hypothesized what might have gone wrong during my execution of these steps. It turns out that I have forgotten a small but very important step: trypsinizing my cells. Without proper trypsinization, HeLa cells continue to adhere to the flask and are unable to be transported into a six-well plates during seeding. The cells that I have obtained from the flask without trypsinization were not live cells to begin with. After troubleshooting, I also wrote down the details into my notebook on what the cells should look like after each step of my experiment. This greatly reduced any doubts I had while re-running the 3rd trial today. The big lesson that I learned through all this is that trial-and-error is what research is all about and that disappointment and troubleshooting are also integral parts of the research progress. We should not only seek to get the data we want, but to also appreciate the lessons that we are able to learn when experiments fail.

TFEB Localization and MK-STYX Expression in HeLa Trials 1 & 2

In order to determine the changes in TFEB (transcription factor EB) localization within HeLa cells in the presence of the pseudophosphatase MK-STYX, HeLa cells were transfected with pMT2 (control), MK-STYX, and F1 (active mutant of MK-STYX). After 24 hours, transfected cells were serum-starved over night (~18 hours) and immunostained for the TFEB, an autophagy regulator. TFEB is normally localized throughout the cell, including the cytoplasm and the nucleus, but its presence in the nucleus would indicate an upregulation of autophagy. After running the first two trials, fluorescent microscopy results indicated a role of MK-STYX in inducing TFEB localization. However, the data for the cells transfected with pMT2 could not be used because most of these cells lysed after immunostaining. At this point, I suspect that the cells might have lysed due to dehydration during immunostaining, being overstressed, or both.