My third week started with me having to make sure that my hematopoietic mouse stem cells completely differentiated into macrophages, which should have taken five days to occur. Once I had the macrophages, I treated them with different ligands (low-density lipoproteins (LDL), lipopolysaccharide (LPS), and dimethyl sulfoxide (DMSO).) The goal of this experiment was to recreate the environment where the expression of a Cyclooxygenase (COX) 2 gene would become over expressed. (The COX-2 gene is induced by the LPS and LDL.) In a real life application, the environment where the cells are kept, is similar to a patient that has atherosclerosis (a cardiovascular disease where macrophages consume oxidized LDL and form plaques in blood vessels). Eventually, to observe any potential changes to the phenotype I was told to use a fluorescent microscope. Hence, from the name, I needed to take the COX-2 protein with a fluorescent that would show the presence of the COX-2 protein in the cells. (By the way, I performed these experiments using a COX-2 knockout and the wild type). The process used to tag the proteins is called Immune Histology. Immune histology uses two antibodies, the primary to bind and detect the presence of the desired protein, COX-2, and the secondary antibody has the fluorescent tag attached to it and binds to the primary antibody. View the figure (figure 1) bellow to see the process on a molecular level. In addition to the fluorescent bound to COX-2, I used another one, DAPI, which binds to the nucleic acids within the nucleus. This DAPI dye allowed me to see the location of the nucleus in the cells in addition to knowing that the microscope was functioning properly.
Figure 1:
Once I made slides containing 8 wells (4 with the antibodies and 4 without), I prepared them to be observed through a fluorescent microscope. At first, my postdoc wanted us to use a confocal microscope (figure 2), but after we attempted using it with another postdoc (my postdoc did not know how to operate the confocal microscope), we learned that it was too time consuming and we ended up using a much simpler fluorescent microscope (figure 3). The second microscope was far simpler to use, and I picked up the process fairly quick. Along with gathering the results (image of results below- Figure 4) of the experiment, me and the other high school volunteers were told to create a 20 minute power point presentation, which would be presented at a symposium on Monday. Unfortunately, our parents were invited. After the symposium, it was the last day for the other volunteers, so we all (postdocs and students) went on a scavenger hunt around the Penn campus. This was very fun, not only because I saw parts of the campus that I probably would not have seen, but it was a nice way for us to end the week. Finally, to top that all off, the lab manager threw a pizza farewell party for all of the volunteers. As for today, I moved my desk closer to my postdoc and they allowed me to have my own instruments (centrifuge, pipette (now I don't have to share), computer, and SPACE). Either way, I am looking forward to next week where we are going to the Penn museum on Tuesday and I have off for the fourth of July!
Figure 2:
Figure 3:
Figure 4:
(DMSO treatment: merge of Nucleus and COX-2 stains)
(Wild type untreated)
(KO untreated)
(LPS wild type treatment)
(LPS KO treatment)
Figure 1:
Once I made slides containing 8 wells (4 with the antibodies and 4 without), I prepared them to be observed through a fluorescent microscope. At first, my postdoc wanted us to use a confocal microscope (figure 2), but after we attempted using it with another postdoc (my postdoc did not know how to operate the confocal microscope), we learned that it was too time consuming and we ended up using a much simpler fluorescent microscope (figure 3). The second microscope was far simpler to use, and I picked up the process fairly quick. Along with gathering the results (image of results below- Figure 4) of the experiment, me and the other high school volunteers were told to create a 20 minute power point presentation, which would be presented at a symposium on Monday. Unfortunately, our parents were invited. After the symposium, it was the last day for the other volunteers, so we all (postdocs and students) went on a scavenger hunt around the Penn campus. This was very fun, not only because I saw parts of the campus that I probably would not have seen, but it was a nice way for us to end the week. Finally, to top that all off, the lab manager threw a pizza farewell party for all of the volunteers. As for today, I moved my desk closer to my postdoc and they allowed me to have my own instruments (centrifuge, pipette (now I don't have to share), computer, and SPACE). Either way, I am looking forward to next week where we are going to the Penn museum on Tuesday and I have off for the fourth of July!
Figure 2:
Figure 3:
Figure 4:
(DMSO treatment: merge of Nucleus and COX-2 stains)(Wild type untreated)(KO untreated)(LPS wild type treatment)(LPS KO treatment)
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