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.

SLX5 and Huntington’s Disease: Second Update

In my last blog post, I spoke about developing a protocol to verify findings from a new data set. This has been my focus since that update.

[Read more…]

The Munching: Experimental procedures and data analysis

smiley punches

[Read more…]