Lauren Donato Week 3 Mason Lab
This week has been busy. I have become a professional multi-tasker and have squeezed in eating lunch at a personal best of 8 minutes (I usually eat when a gel is running or I am waiting for something to incubate). Nobody eats at a set time here, because everyone is on their own schedule, and I'm not sure if a PhD student here ever eats, or ever leaves the lab for that matter. When I come in first thing in the morning (around 8:30ish) I sometimes run to the animal hospital attached to my lab to meet doctor Mason and whichever dog is in that day. This is often the highlight of my day because I get to meet new people and view the clinical side of my project. When these dogs come in (some from the osteosarcoma trial and some from the lymphoma trial) they either are getting vaccinated or receiving a blood analysis. She takes two tubes of blood from each dog and gives them to me to complete a PBMC and freeze down (in my last blog). What this does is freeze lymphocyte samples from these dogs for use later on. I like this part of the morning because I get to see the side of the project that I do not get to experience solitary on my lab bench. On Monday I competed a lymphocyte proliferation assay. This assay is a test used to measure the ability of lymphocytes to proliferate in response to a certain stimuli. This assay is important for our lab because it can be used to measure improvements in immunological function following therapy and to detect the presence of immune responses against specific pathogens. This assay had to be completed in the tissue culture room under a hood and it was extremely vital to be sterile! This is my hood:
| my hood :) |
Something incredibly funny, yet sad happened earlier this week. I spent hours prepping a virus and was on the final step to centrifuge it. This giant centrifuge is a shared centrifuge between various labs in the building. When I went upstairs to retrieve the virus hours later, to my shock the entire centrifuge was missing. It was literally gone. So I ran to show the other lab members, and they were confused as well. Apparently the lab upstairs was moving and took the centrifuge spinning at close to 8,000 G's with them. My virus was still inside, so there is now a missing biohazard on the loose. Dr.Mason thought this was hysterical, but I had to redo the procedure the next day. Another funny occurrence happened on Thursday. I walked in to the lab and on my grad students computer I saw this cupcake sitting there. I asked her (she was working) why it was there and she had no clue what I was talking about. After asking everybody in the lab where this cupcake came from, we never found out. It was like magic.
| magic appearing cupcake |
Anyways, back to research. I grew B cells from another dog this week, in addition to taking care of my old ones. Growing B cells is a lot like having a pet. You need to feed them, wash them, and check on their growth. My B cells continued to proliferate by restimulating with cytokine IL4 and cyclosporin. I did a flocytometry staining of the B cells to make sure what I saw growing really were B cells. Dr mason used the FACS machine and explained to me what the results meant. Within the next 6 days, I had enough B cells to freeze down for later use as a vaccine. When this vaccine is needed for this particular dog, the tumor RNA will be electroplorated into it. This means the membrane will become more permeable and allow the tumor RNA to enter it.
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| process of electroploration |
Then these B cells will go back into the dog and hopefully express and kill / prohibit the tumor. Aside from my B cell lymphoma project, I started a mini cloning project as well. I did my first PCR on Tuesday. The template I am working with is called CD21 and is 3,000 bp. Our lab had a template that was 200 bp long and the purpose of my first PCR experiment was to see if this 200 bp fragment was expressed in the larger one. For this, I had to design forward and reverse primers by looking at a plasmid map. Ordering primers is a cool thing I never had to do before, but a postdoc Josefine showed me how. This experiment had many controls, and I really learned the importance of having these controls to analyze the data from the gel. When I had 11 mini PCR tubes in front of me I also appreciated the concept of labeling, haha. I made a 5X master mix with buffer, forward and reverse primers, water, and dNTP. I then had different templates as controls: no template control, cDNA from T cells, cDNA from B cells, cDNA 8-, and cDNA 8+. I used these controls to test the CD21 gene and also a housekeeping gene gapdh to make sure I didn't get any false positives. I loaded a gel of this PCR.
| our gel station |
This lab loads gels a little different than I was used to. Instead of using tubes, they use a piece of wax paper to mix the sample and dye. It was actually really fun to do it this way. After examining my gel, I found out this small gene was present in the bigger gene. The next step was to amplify this bigger gene. We weren't sure if any of the enzymes we had (taq, vent, or this green mastermix) would cut it correctly, so I set up an experiment to try. I had 3 controls for each enzyme: B cell cDNA, T cell genomic DNA, and no template control. None of these enzymes worked, so the lab had to order a better one, that will hopefully come soon! I did a few mini preps of bacterial samples, and ran a restriction digest, to help out a postdoc on the lymphoma project.
| my bacterial samples (they smelled) |
It went something like this: "have you ever done a mini prep/restriction digest" "yes" " great here's the samples." And so I completed the task *successfully* with no directions. I also got to use a very cool machine following the mini prep that measured the amount of RNA I was able to extract. It was called a nanodrop 1,000.
| this is what the nanodrop data looks like |
| this was the nanodrop |
I also got to do an Elispot assay this week, which was extremely difficult but FUN!!!! The ELISPOT assay is used for monitoring Cell-Mediated Immunity, due to its accurate detection of rare antigen-specific T cells (or B cells) and its ability to display single positive cells within a population of Peripheral Blood Mononuclear Cells (PBMC) *which I spoke about in my last blog*. In this elispot, I was testing to see if B cells were presenting a specific tumor causing antigen. These Elispot plates costs 400 $ each, and are not reusable, so it was very intimidating!!! The plate looks like this:
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| Elispot plate |
The assay is easy to read for results, but takes about 2-3 days. This cartoon below is the basic process of the assay. The spots in the last picture of the cartoon represent one cell responding to the antibody, thus meaning that the cell presented that specific antigen.
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| Elispot Assay |
| using multichannel pipette to "wash" the cells |
This week was great !!
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