Kabir Grewal, UPitt Lab, End of Week 5

I arrived in the lab Thursday morning soaked after walking a mere 60 feet from the bus stop to the lab.  The rain here in Pittsburgh has been relentless, but thankfully, weather reports call for a clear weekend.  Upon arriving to the lab dripping wet from the rain, I began to work on my main task for today which was observing the growth of the TMSC’s/CSSC’s from my project by taking new pictures of them, and then comparing those photos to the ones I took on Monday.  After examining the cells under the microscope, it was clear that there had been growth of the stem cells, and furthermore, they had certainly migrated even more.  Because of this, I was able to take many pictures of the TMSC’s and CSSC’s.  In addition to the main well plate of the stem cells, I also prepared an 8 chamber slide last week cultured with the same TMSC’s and CSSC’s.  The reason I prepared this is that eventually I will perform immunofluorescent staining on these cells using the confocal microscope to test for the presence of certain antibodies.  This time when I took pictures, I also photographed the growth of these stem cells in the chamber slide.  I then took all the photos and downloaded them to my computer where I edited them and started to compare the pictures I took today to the ones from Monday. 

After finishing all of the photography work for my project, I made my way to the confocal microscope room with Ye where we examined TMSC stainings for the presence of the proteins for our shared project. I have spent a great deal of time using the confocal microscope in the past week, so I am starting to feel a bit more comfortable using it. Next week, I will have to use it for my project so it is good I am getting in some practice with it.

On Friday, as it has been for the past week, I awoke to the sound of the seemingly endless rain pounding on the roof of the house.  After trekking to work and arriving soaking wet yet again I started working right away.  Friday was among one of the busiest days I have had in the lab thus far.  First, I examined all of the stem cells I am using for my project under the microscope.   After examining them closely with Dr. Du, we determined that the ones I had prepared in the 8 chamber slide for immunofluorescent staining were ready to be worked with next week. On the other hand, the stem cells I had grown in the 12 well plate had not grown enough for me to perform qPCR, so I will wait and check their growth again on Monday.  I then proceeded to make new medium for the well-plated stem cells, which I had to do from scratch because the batch of cell medium I had made last week had already run out.  Immediately after I finished this, my lab mates and I made our way to a conference room where we heard a visiting doctor from Hong Kong explain his research regarding Axon regulation and regrowth in mammals.  More specifically, his research seeks to further determine the role of a tumor suppressor gene in most mammals called PTEN. When removed it has been notes that axon growth is stimulated in places where the nerve is damaged, which typically does not happen.  I found this lecture especially interesting primarily because I understood a large portion of it thanks to learning about neurons in AP bio last year. 

Immediately after the lecture, I returned back to the lab and quickly ate lunch because I had to begin the next part of my experiment which involves preparing the stem cells from the 8 chamber slide for immunofluorescent staining which I will perform on Monday.  This involved injecting a substance called triton into the stem cells which perforates the cell membranes, allowing the fluorescent antibodies to move in.  In addition, I injected a substance called BSA into the cells which essentially prevents non specific binding of the antibodies which can sometimes occur, and can result in altered data.  I let the BSA sit in the cells for one hour, and then proceeded to inject 2 antibodies, Z0-1 and OCT4 into each of the 8 chambers. On Monday, I will inject a series of secondary antibodies, and will observe the proteins under the microscope.

Since it is my last weekend here in Pittsburgh, I have began to pack up my belongings.  I honestly cannot believe that it has been six weeks already, I feel like I just got here and started to get in the swing of things.  Since it was my last weekend here, I made sure to visit some of my favorite places in Pittsburgh including a Taiwanese Restaurant with the best black pepper beef I've ever had, and Schenley Park. Thankfully the weather reports proved to be accurate; Saturday was a beautiful day and I'm glad I spent most of it outdoors in the city.