Vivian Sun :: Week 5 :: Out with the old, in with the new (project)!
Week 5 overall was not the busiest, but I still learned plenty of useful skills and techniques, important for lab work!
I started the week off with some more familiar territory: PCR! It was not the real-time PCR that we did last week, but rather traditional PCR, a process/method I learned about and was able to practice in Biotech my sophomore year. I was definitely more comfortable with this! I did regular PCR with Monica to genotype mice that various lab members had bred; the DNA samples came from tail snips (literally the ends of the mice's tails) that Monica took from the mice. We ran the results under a machine that had various light settings and that projected the results on a computer screen. I even got a small printed and laminated PCR result sheet! It turns out that 9 of the 20 mice we genotyped were IL-4 receptor alpha flox heterozygous mice, and the other 11 were homozygous for the IL4Ra flox gene.
I then continued analyzing the flow cytometry data from my spleen/heart experiment previously. I cleaned up/fixed some gates, with much help from Snow, and I also learned how to gate for FACS sorting (a way to identify specific cells while the sample is in the machine, rather than gate for each cell type after flow). I continued my data analysis and worked on my EXP poster. A bulk of my time was spent analyzing data and putting it into graph forms.
I also got a new project! I'll be analyzing bone marrow (BM) cells to determine what cells make up the BM, as well as if there are enough monocytes in the BM for Snow to adoptively transfer into other mice. Snow had previously shown that the spleen was dispensable in the development of EAM, so she was curious if monocytes could be taken from the BM instead of the spleen during EAM in the donor mouse and then transferred into the recipient mouse. However, the BM is really small, so she was also interested in determining how many cells could be obtained. My role in this was to run a small pilot experiment to determine the number of and the composition of the BM cells.
The BM project, however, has to wait until the Thursday of my last week - we need to wait for the mice to be at a certain stage of EAM, and that won't happen until next week at the earliest.
While I waited, however, I did some more data analysis of the results in Excel. I also wrote up a BM protocol (procedure) in anticipation of my BM experiment, as well as began preparation for my presentation to the entire lab (which will take place on my very last day of lab, in week 6).
I learned how to make Giemsa staining slides. Giemsa staining is used to look at the morphology of cells and makes it easier to identify or even just look at various types of cells under a microscope. I got to use an Olympus Camera Microscope, which works like a regular microscope but is also attached to a computer; you can essentially take digital photos of the slide as it looks under the microscope! The cell culture I took cells from was 1.5 weeks after I made the initial culture. The rounder blobs that are lightly stained blue are most likely macrophages; the dark blue dots are the nuclei. The cells with three connected dots are neutrophilic cells. We weren't expecting to see as many macrophages as we did, which was interesting!
I also sorted through the mouse and human antibodies with Katie - inventorying one box of 81 antibodies, as well as organizing them alphabetically, took an hour! There were about 12 boxes we had to sort through, and we managed to sort all of them. The antibodies were not very neatly organized because people took the ones they needed without necessarily putting them back and the inventory list was in dire need of updating, so Katie and I decided to take on the challenge. My PI joked that somebody's retirement fund was used to buy all the antibodies for the lab! (They can be really expensive, several hundred USD for one small tube.)
I started the week off with some more familiar territory: PCR! It was not the real-time PCR that we did last week, but rather traditional PCR, a process/method I learned about and was able to practice in Biotech my sophomore year. I was definitely more comfortable with this! I did regular PCR with Monica to genotype mice that various lab members had bred; the DNA samples came from tail snips (literally the ends of the mice's tails) that Monica took from the mice. We ran the results under a machine that had various light settings and that projected the results on a computer screen. I even got a small printed and laminated PCR result sheet! It turns out that 9 of the 20 mice we genotyped were IL-4 receptor alpha flox heterozygous mice, and the other 11 were homozygous for the IL4Ra flox gene.
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| the traditional PCR results |
I then continued analyzing the flow cytometry data from my spleen/heart experiment previously. I cleaned up/fixed some gates, with much help from Snow, and I also learned how to gate for FACS sorting (a way to identify specific cells while the sample is in the machine, rather than gate for each cell type after flow). I continued my data analysis and worked on my EXP poster. A bulk of my time was spent analyzing data and putting it into graph forms.
I also got a new project! I'll be analyzing bone marrow (BM) cells to determine what cells make up the BM, as well as if there are enough monocytes in the BM for Snow to adoptively transfer into other mice. Snow had previously shown that the spleen was dispensable in the development of EAM, so she was curious if monocytes could be taken from the BM instead of the spleen during EAM in the donor mouse and then transferred into the recipient mouse. However, the BM is really small, so she was also interested in determining how many cells could be obtained. My role in this was to run a small pilot experiment to determine the number of and the composition of the BM cells.
The BM project, however, has to wait until the Thursday of my last week - we need to wait for the mice to be at a certain stage of EAM, and that won't happen until next week at the earliest.
While I waited, however, I did some more data analysis of the results in Excel. I also wrote up a BM protocol (procedure) in anticipation of my BM experiment, as well as began preparation for my presentation to the entire lab (which will take place on my very last day of lab, in week 6).
I learned how to make Giemsa staining slides. Giemsa staining is used to look at the morphology of cells and makes it easier to identify or even just look at various types of cells under a microscope. I got to use an Olympus Camera Microscope, which works like a regular microscope but is also attached to a computer; you can essentially take digital photos of the slide as it looks under the microscope! The cell culture I took cells from was 1.5 weeks after I made the initial culture. The rounder blobs that are lightly stained blue are most likely macrophages; the dark blue dots are the nuclei. The cells with three connected dots are neutrophilic cells. We weren't expecting to see as many macrophages as we did, which was interesting!
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| a 20x magnification of the Giemsa-stained cells. |
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| a 40x magnification of the Giemsa-stained cells. |
I checked on my cell culture one more time at the end of the week to see if there was any contamination in the wells. Luckily it looked fine; no contamination for two solid weeks was definitely a great result!
At the lab meeting at the end of the week, Taejoon presented on his previous research, conducted at the University of Michigan. He focuses on neonatal hearts and congenital heart block, in which the maternal antibodies enter the fetus through the placenta and injure the fetus' heart.
Over the weekend I went to a local arts festival called "Artscape." It's the country's largest free arts festival, and I was lucky enough to be able to visit! There were three or four streets lined with tents full of vendors selling their wares, be that arts or clothing or various household items; there were also plenty of food trucks and restaurants selling their food at the festival, too. There were three or four different stages with performing musical guests! It was tons of fun.
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| the cell culture is clear, meaning that there's been no contamination. The two empty wells on the left are the wells we took the cells from for Giemsa staining, but those two were also similarly rose-colored and clear, just as these four wells with media are. |
Over the weekend I went to a local arts festival called "Artscape." It's the country's largest free arts festival, and I was lucky enough to be able to visit! There were three or four streets lined with tents full of vendors selling their wares, be that arts or clothing or various household items; there were also plenty of food trucks and restaurants selling their food at the festival, too. There were three or four different stages with performing musical guests! It was tons of fun.
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