So everything got deleted as I was writing, so I have to rewrite everything on Thursday, 8/11. Not everything will be accurate since I am not writing on the day of. I'll try my best though.
Victor is planning on doing another experiment with me. Originally, it was supposed to be the time lapse experiment, but because of tricky scheduling, he decided not to do it. Instead, we are doing an experiment with RELA, a transcription factor in the NF-kB family. He wants to find the protein concentration in cells and how much translocates into the nucleus. RELA is generally in the cytoplasm of the cell, bound by specific receptors inside the cell. When perturbed, the RELA break away from the receptors and go into the nucleus to regulate transcription. We are not perturbing the cells with any external stimuli, such as TNF, so we can accurately image the RELA concentrations in the cells. Victor also wants to fix the cells so they do not escape the traps, like they've been doing in my TNF experiments.
I attended a thesis defense for a graduate student named Stacey Lawrence. It was at the Kline Geology building behind the Peabody. She presented her research on how G proteins affect the immune receptors/response (I think, I forgot since I accidentally deleted everything I wrote. I also fell asleep halfway through the presentation, whoops). The closed Q&A session with the thesis committee, which usually go on for about 15-20 minutes, according to every grad student I asked, lasted for over an hour. The food from Thali was worth it, though.
I went back to lab and started on my experiment. I did the first stain of NF-kB antibodies. The antibodies will attach themselves to the RELA. I also "fixed" the cells, meaning that I killed them so they become rigid. It's not apoptosis. I also had to permeabilize the cells to allow the stain to get inside of the cell. I used ice-cold MeOH to permeabilize. Don't know how it works, never asked (according to grad students, as long as it works, you don't ask questions). I then put the device with the cells in a container and into the fridge to leave overnight. Victor wrapped the container in parafilm to avoid evaporation. This would cut off the exchange of gases, but since the cells are already dead, it does not matter.
I started work in the lab early, and by early, I mean I started as soon as I walked in. I applied the 2nd stain to the cells. The stain was called Alexa 488, a molecular probe that attaches itself to antibodies. The devices had really good trapping efficiency, with over 80% of the traps filled with at least one cell. Since the devices were in the fridge overnight, there was condensation on the devices, so it was somewhat difficult to see the channels at first. I also added a Hoechst stain so I can identify the DNA and the nucleus in the cells.
There was a lab meeting today where Victor Bass, another graduate student, was presenting his progress on his research. He was studying TNF and LPS stimulation. I did not really understand everything because of the graphs, since they always mess me up. Andres was supposed to present a paper, but he forgot, so it was a short meeting.
After the lab meeting, I imaged my cells in the devices. I took 10 pictures of at least 15 cells, so at least 150 cells, max 200. HIV was activated in some cells, making the cells appear as if they're from the Matrix. Since they were so bright, I could not see the RELA concentrations in the cells, so I could not quantify those. I could only quantify the cells without active HIV but had RELA, which was all of the cells. I have to analyze the intensity of the light, or the grayscale intensity, the standard deviation of the light intensity, and the integrated density. The integrated density is the area times the average mean gray value, or light intensity. The integrated density is therefore the light intensity of the entire cell. I will have to analyze each cell individually since the data also quantifies the cells with activated HIV, so that's a pain.
I met with Kathryn, my P.I., in the morning to discuss how things have been going in the lab. I told her about my RELA experiment and that I had a great time learning and doing experiments in the lab. We talked about if I could come work in the lab throughout the year, but she did say scheduling would be tricky. I also just realized that I will be busy throughout the year with college classes and extracurriculars, so I will have to tell her that. She is planning on taking another high school intern next year, but also wants me to return so we can learn together. I can help the high school student as well as learn in the lab. Kathryn also told me about Victor's paper on how RELA regulates gene expression since it's a transcription factor, and how Victor is trying to determine a way to predict how RELA will behave. They plan to use this work to treat disease and things like that.
I also went with Victor to West Campus since he had to use some tech there for his experiment. Victor asked if I wanted to go with him on Tuesday, so I said yes. We ate tacos at the cafeteria and met with another graduate student there. The microscope Victor used was extremely powerful, with magnification capabilities of up to 100x magnification. Victor was going to use the microscope for a long time, so I left at 3:30 because I needed to go home.
I got gifts for Victor, Laura and the professor. I also got donuts for everyone, but only Andres and Victor Bass showed up to lab. I just stayed in lab and worked on my personal projects.