Peddie EXP 2017

This past week was my last in the lab.  In the middle of last week, I continued to work on immunofluorescent staining.  This took a couple days to finish because the antibodies need to be left on the staining slide overnight.  I added 2 more antibodies: DAPI which stains for nuclei, and another general stem cell marker called ­­­­CD271.  When I originally observed the stem cells under the confocal microscope, I did not see what I was expecting to; instead, I saw blurry colors that were not vibrant at all.  This made me quite nervous because this is essentially what I had been working towards for the better part of six and a half weeks.  After looking at the stained cells under a regular microscope, we discovered that in each of the 8 slides, the cells had grown primarily around the outside of the slides and sparsely in the middle region.  With this new knowledge, plus the help of Dr. Du, we were able to take some great pictures, some of which are show...

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 con...

In my previous blog post, I discussed the work I did last Thursday and Friday in which I made a new growth medium for the co-cultured TMSC’s/CSSC’s and lysed BCE cells. This brings me to the work I completed on Monday, Tuesday, and Wednesday.  When I arrived at the lab on Monday, I immediately checked the stem cell's growth.  Overall, they  looked good. As seen in the pictures below, some of the big clumps of cells (which are the TMSC’s/CSSC’s) have anchored themselves on the bottom of the wells, and have begun to migrate towards the extra cellular matrix of the lysed BCE cells.  In most of the wells, however, the stem cells did not seem anchored or migrating yet; this is ok because it is still early, so by the end of the week they should be.  After this, I changed the growth medium of the cells.  This can be a tedious process because since many of the stem cells are still free floating in the growth medium, they can be mistakenly sucked out in the pro...

I ended my last blog post with writing about when I injected TMSC’s and CSSC’s into  well plates with  the extracellular matrix of bovine corneal endothelial cells (BCE cells) , which brings me to the work I completed last Thursday and Friday. Last Thursday was a busy day for me.  After arriving at the lab at the usual time of 9:30, I checked the growth of the newly injected stem cells mixed with the extracellular matrix of lysed BCE cells. As expected, the stem cells had not attached to the bottom of the well plates, and were free floating in the DF10 growth medium. Today, my task was to make a new growth medium for these cells, as well as create the variables for the experiment.  The new growth medium I made comprised of a glucose-rich base, fetal bovine serum (FBS), as well as ROCK inhibitor (short for Rho Kinase inhibitor). The purpose of this inhibitor is to prevent excessive apoptosis among the cells, and promote the cells to grow and retain their struc...

As it turns out, my previous blogs didn't save/upload correctly, so here I am writing my first blog going into my fifth week in the lab. The first two weeks in the lab involved a lot of basic training, so there is not too much to write about there; I will mainly focus on the third and fourth week. Because of this, this post is going to be a bit lengthy, so bear with me.   Fortunately, however, this bad luck is not at all representative of how my first four weeks in the Du lab at the University of Pittsburgh have gone thus far.  The Du Lab is a small lab (8 people total including myself) in which there are two main focal points; the first is harnessing the regenerative capabilities of stem cells to try and treat the dysfunctional trabecular mesh work that causes Glaucoma, and the second being trying to find an ethical yet effective way to regenerate lost corneal endothelial cells. For the first 1-2 weeks, most of my days were spent learning my way around the lab/finding all ...