Vivian Sun :: Week 2+3 :: Hands-on Work and Protocols
Week two, because I got my animal training approved and all my paperwork done, was when things really began to get interesting! Just a quick overview of the past two weeks: I dissected a mouse, helped Snow perform sacrifices and heart harvests, analyzed cardiac tissue using flow cytometry, and learned about projects that'll begin next week. I was also able to visit the Inner Harbor and Washington DC this weekend, a due recess after a busy workweek. I wrote two protocols with Snow, handled some more mice, analyzed a lot of flow cytometry data, and even got my own mini-project (in addition to helping Snow with hers)!
Things got interesting on Monday. Snow was sick, so I was shadowing Paul and watching what he was doing. One of the mice he was immunizing in the animal room suddenly died in the hood (the sterile area that people work with live mice in). Katie and I took it back to the lab to dissect it to see if we could figure out what went wrong. When we cut it open, we could see a small white bulbous area near the lymph nodes on the right side (the mouse equivalent of the underarm area), where Paul had injected an emulsion the previous week. The injection is subcutaneous, meaning that it is right below the skin, just like a TB test. We hypothesized that perhaps he had injected too deep and the emulsion went into the chest cavity, which could have caused the abrupt death. The only strange thing was that we couldn't see the emulsion he injected just 15 minutes prior, near the left lymph nodes. We weren't able to figure out what went wrong, but it was an interesting dissection. I got a preview of how harvests work!
After Monday: The sacrifices done this past week were for a small pilot experiment Snow designed for my PI's eosinophil grant proposal. While I wasn't able to handle the mice myself, I was able to watch and learn a lot from it. The experiment's purpose was to identify the cells that express the receptor for interleukin 13 alpha 1 (IL-13Ra1) during EAM (experimental autoimmune myocarditis, a mouse version of human myocarditis). Because it was a pilot experiment, only six mice were involved - it was to simply test the waters and determine if the project was worth pursuing.
A lot of preparation is required to sacrifice mice and harvest their hearts (another way of saying "euthanize the mice and extract their hearts for analysis"). Such prep is done the day before the sacrifice due to the amount of time required to get or create the necessary materials - it minimizes stress on sacrifice day.
After bringing the pilot experiment mice up to the lab from the animal room, Snow and Katie put the mice to sleep with avertin. Then, they collected blood samples by performing either a cheek bleed or a retro-orbital bleed. The blood is centrifuged and its serum analyzed for antibodies, but this is not hugely important. About half to two-thirds of a 1.5mL vial of blood was collected, which a lot of blood for a small mouse, but the mice are to be sacrificed almost immediately after.
In order to harvest the hearts, it was necessary to euthanize the mice. After doing so, Snow perfused the hearts to wash out the circulating red blood cells so that they would not interfere with the collection of heart cells. She then cut the aorta and took out the spleen, which is crucial because of its function as an immune system organ and its abundance of immune cells (it can be used as a comparison). She perfused the right ventricle again and weighed both the heart and the spleen, then mashed up the heart. She cut a fourth of the spleen and put both organs in a C-tube and a well with 1X HBSS, respectively (Hank's Buffered Salt Solution, an isotonic solution for cells so that they do not lyse or swell and burst). After essentially blending the heart, adding enzymes, filtering, washing, and vortexing, the heart cells are suspended in a single cell suspension and set aside for analysis with flow cytometry.
Flow cytometry is, in essence, a single file line of cells suspended in solution and put into the flow cytometer. One by one, cells are sucked up into the machine and lasers are shot at these cells. Each cell has different markers/antibodies, so they each emit different wavelengths of light when hit with the laser, which the machine detects. They do this because the antibodies on the cells have fluorochromes, a dye that is assigned to a specific marker that can be read by the machine and used for analysis.
Over the July Fourth weekend, I first stopped by Baltimore's Inner Harbor. I also visited Washington DC and revisited the National Gallery of Art! I also went to some smaller museums I hadn't been to in the past, namely the Hirshhorn Museum and the Holocaust Museum. It was really hot, but the museums had awesome exhibits. Ai Weiwei, for example, had an exhibit called Trace at the Hirshhorn Museum that I really wanted to see - it featured activists who had been jailed or otherwise punished for their efforts. It involved a thought-provoking wallpaper and Lego portraits of the aforementioned activists, as well as interactive monitors that gave a brief biography of each activist. It was really eye-opening. I also got a chance to visit the National Aquarium in Baltimore on July 4th. It was huge and I loved everything in it, especially the jellyfish exhibit!!
Things got interesting on Monday. Snow was sick, so I was shadowing Paul and watching what he was doing. One of the mice he was immunizing in the animal room suddenly died in the hood (the sterile area that people work with live mice in). Katie and I took it back to the lab to dissect it to see if we could figure out what went wrong. When we cut it open, we could see a small white bulbous area near the lymph nodes on the right side (the mouse equivalent of the underarm area), where Paul had injected an emulsion the previous week. The injection is subcutaneous, meaning that it is right below the skin, just like a TB test. We hypothesized that perhaps he had injected too deep and the emulsion went into the chest cavity, which could have caused the abrupt death. The only strange thing was that we couldn't see the emulsion he injected just 15 minutes prior, near the left lymph nodes. We weren't able to figure out what went wrong, but it was an interesting dissection. I got a preview of how harvests work!
the lab desk where sacrifices are performed and tissue samples collected
the styrofoam platform used to dissect mice and harvest their hearts/spleens
After Monday: The sacrifices done this past week were for a small pilot experiment Snow designed for my PI's eosinophil grant proposal. While I wasn't able to handle the mice myself, I was able to watch and learn a lot from it. The experiment's purpose was to identify the cells that express the receptor for interleukin 13 alpha 1 (IL-13Ra1) during EAM (experimental autoimmune myocarditis, a mouse version of human myocarditis). Because it was a pilot experiment, only six mice were involved - it was to simply test the waters and determine if the project was worth pursuing.
A lot of preparation is required to sacrifice mice and harvest their hearts (another way of saying "euthanize the mice and extract their hearts for analysis"). Such prep is done the day before the sacrifice due to the amount of time required to get or create the necessary materials - it minimizes stress on sacrifice day.
After bringing the pilot experiment mice up to the lab from the animal room, Snow and Katie put the mice to sleep with avertin. Then, they collected blood samples by performing either a cheek bleed or a retro-orbital bleed. The blood is centrifuged and its serum analyzed for antibodies, but this is not hugely important. About half to two-thirds of a 1.5mL vial of blood was collected, which a lot of blood for a small mouse, but the mice are to be sacrificed almost immediately after.
In order to harvest the hearts, it was necessary to euthanize the mice. After doing so, Snow perfused the hearts to wash out the circulating red blood cells so that they would not interfere with the collection of heart cells. She then cut the aorta and took out the spleen, which is crucial because of its function as an immune system organ and its abundance of immune cells (it can be used as a comparison). She perfused the right ventricle again and weighed both the heart and the spleen, then mashed up the heart. She cut a fourth of the spleen and put both organs in a C-tube and a well with 1X HBSS, respectively (Hank's Buffered Salt Solution, an isotonic solution for cells so that they do not lyse or swell and burst). After essentially blending the heart, adding enzymes, filtering, washing, and vortexing, the heart cells are suspended in a single cell suspension and set aside for analysis with flow cytometry.
Flow cytometry is, in essence, a single file line of cells suspended in solution and put into the flow cytometer. One by one, cells are sucked up into the machine and lasers are shot at these cells. Each cell has different markers/antibodies, so they each emit different wavelengths of light when hit with the laser, which the machine detects. They do this because the antibodies on the cells have fluorochromes, a dye that is assigned to a specific marker that can be read by the machine and used for analysis.
a flow cytometer. the test-tube samples go in the small indent in the lower right corner of the machine.
the inside of a flow cytometer
When obtaining data from the flow cytometer, we had an issue where one of the compensation markers we used wasn't showing up on the screen and data wasn't being collected. (Compensation is a way to compensate for the background noise/wavelengths of light that may overlap with other wavelengths; the compensation results are used as a baseline to identify which emission spectrum is the actual emission spectrum and which one is background noise from previous samples, etc. It's like using noise-cancelling headphones to compensate for the noisy environment around you so that you can hear your music more clearly.) Without compensation, the data collected is useless since it's unknown if the data is accurate or if it's a false positive caused by background wavelengths/noise. Snow borrowed Hee Sun's compensation, which (thankfully) worked and we were able to continue data collection!
After she obtained all the data, she used FlowJo to analyze it. Snow taught me how to gate out unnecessary cells and how to select for the cells that we actually want. Gating is a way to filter out the unwanted data and focus only on the ones we want. It involves making a shape (box, circle, polygon, ellipse, etc.) around the desired cells - after you make that shape, another window pops up with just those cells in a graph. You can identify the cells you want by utilizing the x- and y-axes labels (usually a type of antibody/marker specific to a cell type). For example, you have a regular coordinate grid, only the first quadrant. Split that first quadrant into four squares. The bottom left square is negative for the antibody labels on both axes (meaning that any cells in that corner do not have either of those antibodies), and the bottom right is negative for the y-axis label, positive for the x-axis label. The top right is positive for both antibodies (meaning that any cells appearing there would have both antibodies), and the top left is negative for the x-axis, positive for the y-axis antibody. By taking a broad category of cells and narrowing it down using specific antibodies, you can strategically gate and identify cells. You need to choose your markers based on what cell expresses what.
After that, I worked on two protocols for Snow's future projects. I also went down to the animal room with her and helped her sort out/organize her multitude of mice cages (it's necessary to maintain and check up the cages at least once a week to prevent overcrowding and to make sure that the mice are generally healthy, not fighting, etc.). I practiced picking the mice up by the tails and gripping them by the scruff of their neck so that they will not move or bite when they're being immunized. I also practiced differentiating between the genders, a crucial skill since male mice are more susceptible to severe EAM (which is necessary for our projects!) than females are.
There was no lab meeting at the end of the second week due to the holiday weekend, so I worked on my protocols at my desk instead!
a view of my desk. I sit next to a really fancy microscope! (I'm not sure if I'll be able to learn how to use it)
a really nice view of the Inner Harbor!
Ai Weiwei's portrait was the first thing I saw when I entered the exhibit at the Hirshhorn Museum.
Lego portraits on the ground, surrounded by the 360-degree wallpaper
the wallpaper is called The Plain Version of the Animal That Looks Like a Llama but Is Really an Alpaca; it features handcuffs, security cameras, alpacas, and the Twitter logo.
the first thing I saw when I entered the National Aquarium: A giant underwater reef!
My third week began on Wednesday, July 5th. I continued working on the two protocols and helped Snow check up on her mice after the long weekend. I also learned how to ear-tag the mice, which is exactly what it sounds like: I pierced their ears with a very small metal tag so that we could identify which mouse is which. I learned how to use Excel and Prism to analyze the numerical data obtained after gating with FlowJo after flow cytometry. The math was easy, but the concepts and thinking required a bit more effort - I realized that biological analysis of data is radically different from the math in math class (no pun intended).
Every Friday has a lab meeting, but this one was a special meeting - it was the myocarditis group meeting. It was a time for group feedback and suggestions/constructive criticism on everyone's protocols. Everyone presented their current protocols, and Snow presented the ones I wrote with her!
Wrapping up the week, I got my own mini-project! As it turns out, I will be helping Snow with the experiments she'll be running in the next few weeks, but I won't be able to see the outcomes (I have to leave before the experiment is finished). There's also a bit of downtime in the middle of each project since it requires waiting for the mice to develop EAM to a certain stage before we can sacrifice them and harvest their hearts/spleens for analysis. As such, my own project will help Snow out in the long run, but more on that next week (when I have a clearer idea of what I'll be doing). However, procedurally, I'll be performing tail vein injections on the mice and also will run flow cytometry, do data analysis, and present on my findings at the "Summer Student Presentation" meeting at the end of my time at the lab.
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