Rachel Henning ’15, Becker Fellow in Research
On Monday I ran another BCA protein assay to compare the protein concentrations of unstimulated and stimulated exosomal protein from dendritic cells. My results definitely improved from the last time I ran A BCA protein assay. I also fixed some slice cultures with methanol which tends to make better imaging. However, I went to mount the slices onto a slide I learned that the methanol dehydrates the slices a bit and they were very tricky for me to mount! So my job is to figure out which cells these exosomes, which are only like 100 nm, are traveling to. Partially since the exosomes are so small I tag the exosomes with QD-CD63, which fluoresce red, and I stain certain cells within the slice cultures. I then look at the slices under a microscope and see which cells the red dots went into. The QD-CD63 are super sensitive to light so I have to keep the slices in the dark as much as possible so I hide the slices under a box.
Tuesday I stained for oligodendrocytes and when I looked under the microscope it appeared that the exosomes had entered into the oligodendrocytes. But since the microscope in the lab can’t look at different planes, it is hard to tell if the exosomes are in the oligodendrocytes or just the surface. The only way to know for sure is to go to the confocal microscope, which can look at the cells from all different planes. But my staining worked which is always a good thing!
Our standards for the BCA protein assay were getting low so I got to make new ones. As you can see in the picture, the intensity of the purple varies, which is due to variations in protein concentration. My new standards are the lower two rows and the old standards are the upper two rows.
Wednesday I stained some slice cultures for microglia and put some tagged exosomes in! This is the final product!
So the green stuff is microglia cells and the red are the tagged exosomes. The orange and yellow areas are places where the exosomes went into the microglia! Success!!!!
On Thursday my program has it arranged that we get to have lunch with mentor students. The mentor students are students who are either in medical school, graduate school, or both! We got to chat with them and ask any questions we had! It was a great opportunity to hear a bit more about the Pritzker School of Medicine here at the University of Chicago. Apparently here you don’t receive grades your first 2 years of medical school! Interesting! I also got to learn how to do an SDS-page gel, which separates proteins based on their size. I was then able to take the separated proteins from the SDS-page gel and transfer them to a western blot.
On Friday I took the western blot from Thursday and tagged Alix proteins, which if present will help us confirm that we have exosomal protein. We had a shorter day because there was a BBQ for everyone who’s here over the summer that evening. It was great to get to talk to other summer program students as well as doctors and researchers.