Deja Cunningham, Week 5, Presenting and Preparing Plates!

As my second to last week at Duke has just finished, I can definitely say that this has been the best experience I’ve ever had working in a lab. On Monday, I presented at our weekly lab meetings on the progress Elizabeth and I have made on our in vivo experiment. I was at first nervous to present because I didn’t want to mess up or have a low quality PowerPoint compared to the other members of the lab, but after I finished I had a feeling of relief. My PI and other members of the lab congratulated me on a great presentation. This made me feel really good.

Tuesday, was a very busy day. In the morning, Elizabeth and I created the plates to culture our Bone Marrow Derived Macrophages. To do this, I first sacrificed one mouse with CO2 and then did a thoracotomy to ensure that the mouse could not breathe again. Then I cut out the femur with the fibula attached to it. With a syringe, I flushed out the bone marrow with PBS (Phosphate Buffer Saline) into another plate. At times, it was hard to get the needle into the small hole of the bone without your hand shaking, but after a while, I got the hang of it. Following that, I made a single cell suspension by pipetting the mixture up and down and then putting it through filter paper into a clean tube. I centrifuged the sample, which caused a pellet to form at the bottom containing white and red blood cells. Since we aren’t testing the red blood cells, I added hemolysis buffer to the sample in order to kill the red blood cells. Then, I centrifuged the sample again to create a pellet of only white blood cells at the bottom. I discarded the liquid at the top and then re-suspended the pellet in RPMI-MCSF, which contains the medium (has antibiotics to prevent bacteria growth and proteins which make the cells happy) and the growth factor for macrophages, M-CSF. I put the solution on five dishes and then in the incubator to sit for 6 days.

After preparing the plates of the BMDM, we began setting up our plate for another OPN ELISA. This time we had many wells because we were trying to compare the concentrations of OPN in the WT unstimulated and WT CNT stimulated mice at 6hr, 24hr, and 3days. We wanted to see that in the WT CNT stimulated mice there was much more Osteopontin than in the WT unstimulated. Unfortunately, we did not see this. Our data showed no difference in the protein levels in the WT unstimulated compared to the WT CNT stimulated mice. However, we can at least say that over the course of time there are higher levels of Osteopontin.  

During the week, our lung samples we sent to the Duke CORE facility to be embedded in paraffin finally were finished. To prepare for another H&E staining, I used the cryostat microtome to slice 7μm thick slices of each sample. This took a long time because we had 11 samples and I needed to make two sets of slides with two lung slices on each slide. Soon after I began the H&E staining on one set of the slides and as I was dipping the slides in xylene to deparaffinize the slides, a substantial amount of the actual lung samples came off too. I was startled at first and thought that it would eventually stop, but when it didn’t I knew I needed to tell Elizabeth. We tried to think of what could have been the problem and the only thing we could think of was because the slices were too thin. So, I had to RESLICE two more sets of the sample AGAIN, but this time making the slices 10μm thick (image to right). It was super tedious, but luckily I got through it.

Since the primers for the five genes I said I planned to test in qPCR came in as well, Friday I set up a plate for them. The five genes I was testing were IL-6, IL-33, PPARγ, Edn1, and IFNγ and I wanted to see the expression levels in the WT unstimulated and WT CNT stimulated mice at 24hr. Unfortunately, the data for this didn’t look so good either. Many of the samples showed very low levels of mRNA for a specific gene. 

On the Weekend, My mom and I explored many of the food options Durham has to offer. We went to a popular wing place called Heavenly Buffaloes twice, which is superrrr good. We also went to Dames chicken and waffles. It has to two best things ever. CHICKEN and WAFFLES. Whoever created this concept is brilliant. This picture is a solid representation of what I had (image to right). They even have these whipped butters called schmears that come in all different flavors. I had a vanilla-almond schmear. Let me tell you. SO GOOD. Especially with maple syrup.

We in addition went to Bull City Burger, which is supposed to have the best burgers in all of Durham. Now, I’ve tried many burger places since I love to eat and personally I thought that they burgers were good, but not the best I’ve ever had. To top it off, we went to Small Cakes for cupcakes and Sugarland for gelato and cupcakes. It was my first time having gelato and I thought it was pretty good, but the cupcake I had from Sugarland was DELICIOUS. Oh yeah and we even went back to Elon to have a taste of those amazing tacos for the last time.

I can’t believe that I only have one week left. I will for sure remember this experience!!