WISE journal entry #23

Name: Leah Gerald

Entry: #23 (5/15/2012)

WISE Week:12

Written on Date:5/15/2012

Mentor: Sagar

today was a presentation day for sagar and a handful of other students in the lab. it was a chance for them to present what they've been working on and whatever results they've found. my mentor had a poster that talked about what hes working on, and after I went back to the posters several times because today they were trying something very different. Sagar explained to me how in clumps of cells, the outer cells have  access to oxygen, but the inner cells don't, therefore they are in a hypoxic condition, and they cannot reach oxygen, so in cells that are in this condition, they have a gene that expresses this protein called Hif1 alpha. Hif1 alpha allows these cells another means to survive as a substitution for oxygen. they're not sure in which area in the chain of command where hif1 alpha is expressed, so they knocked down several different levels using antibiotics. they weren't able to do much today though except to quantify the cells that had the antibiotics in them and make sure enough viable cells remain. they also did something today they call licing the cells. the purpose of licing the cells is to open them up, which is how they extract the hif1 alpha protein, which is how they can see how much expression of it is going on within the cells

WISE journal entry #22

Name: Leah Gerald

Entry: #22 (5/10/2012)

WISE Week:11

Written on Date:5/13/2012

Mentor: Sagar

today, sagar was planning on using a flow-cytometry machine. we needed to use this machine because they needed to look at specific protein the cells produce (one in particular, but I cant remember the name) that will help them classify/ know/ find stem cells in the cancer cells. this protein is found in cells that self regulate (a stem cell-like behavior), which is why they are trying to spot out the cells that produce it. the first part of the day was spent quantifying the cells, because we needed at least half a million cells for the machine. after they were  measured out and spun down a couple times, we added antibodies in the cells which should attach to the protein of interest and the machine will light up the cells with the antibodies attached, thus proving if or if not the cells express the protein of interest. the cytometer does this basically by sorting the cells. it takes them all, one at a time and sorts them into cells populations. with this information, the machine created several graphs which had many clustered areas that my mentors sectioned and noted the differences in the different samples. there was a control, and for all the other samples, if it differed from the control, that showed how much it expressed the protein of interest

WISE journal entry #21

Name: Leah Gerald

Entry: #21 (5/9/2012)

WISE Week:11

Written on Date:5/13/2012

Mentor: Sagar

today, the first thing we were to work on was an RNA isolation procedure, which I have never seen before. basically, the RNA isolation procedure is, as the name denotes, to isolate RNA. to do so, we had to pour a bunch of buffers through the RNA and spin it down several times to wash it of contaminants so when looking at the RNA later, nothing will get in the way, like DNA and contaminants. cells don't always use all their genes, so looking at the RNA shows us which genes they are ACTIVELY using. this procedure didn't take very long, and for the rest of the day, there were a lot of cells that needed to be re-fed new media, so I spent the most of the day in the cell culture room doing just that. I probably added media to 20-30 flasks. also, Sagar showed me some recently acquired xeno-grafted cordoma cells. they are called this because foreign cordoma cells were placed in a mouse, and then extracted. because the cells were placed in a body, they are mixed up with many other cells, so the plate with the xeno-grafted cordoma cells had a mixture of more than just cordoma cells.

WISE journal entry #20

Name: Leah Gerald

Entry: #20 (5/2/2012)

WISE Week:10

Written on Date:5/6/2012

Mentor: Sagar

today, I had the hood to myself for the first time ever! Sagar had set aside a time and some lung cancer cells that had become too confluent for me to play with that day. I cleaned the station, got out what I would need for my lab, and got to work. I was to take the cells out, count them, dilute them properly, and replate them in 6 different dilutions. first, to get the cells out, I washed them with PBSO, which helps remove calcium, which slows down the trypsin. then I added the trypsin and waited 5 minutes for the trypsin to disconnect all the cells from the bottom of the plate. then I added some media to the mixture to help calm the trypsin down, and spun the cells down in the centrifuge. I then removed the trypsin and media, and mixed the cells with some new media. then I took a small section of this new mixture and counted the cells in the cell counter. I found out how many cells were in one mL, and then had to create 6 different dilutions so that I would have 1 million, 500,000, 50,000, 10,000, 1,000, and 100 cells in each separate well. this part proved very difficult for me, because the mental math was kind of rough for me, and also I never did very well with dilutions in math. so I messed that part up several times and had to redo my dilutions more than once. in the end, I'm not sure I even got it right, but the fact that I did it was enough I guess

WISE journal entry #19

Name: Leah Gerald

Entry: #19 (5/1/2012)

WISE Week:10

Written on Date:5/6/2012

Mentor: Sagar

today, I spent the first part of the day researching fixation, immuno-florescence, and H & E staining, and their connection, because earlier that week, my mentor had recieved some mouse brains from germany which he was staining with H & E. basically, first they had to fix the brains, which causes them to not deteriorate. then, once they are fixed, they stain them with H &E, which colors the nuclei of the cells blue and the other parts red. after this, I went into the hood with Nate, who was replated cells once again, but something else exciting was going on that day, so I was between two things. in the other room, some of the other lab members were preparing to inject some mice with some cancer cells, so my mentor told me I could watch if it interested me. I went to and from the mouse surgery, because It freaked me out a little bit. they pinned the mouse down, cut their skin open, counted down a couple of ribs to the point in the mouses body where its heartbeat was the greatest and injected the cells. at one point, we could see the mouses beating heart under the muscles. I went back to the hood several times because I couldn't watch the whole process. when I did, there were some issues because Nate was replating these cells that showed an irregularity I couldn't quite note.

WISE journal entry #18

Name: Leah Gerald

Entry: #18 (4/24/2012)

WISE Week:9

Written on Date:5/6/2012

Mentor: Sagar

today my mentor was planning on performing a transduction, but unfortunately we never got to it. a transduction and a transfection have basically the same results, but the processes and results differ slightly. with a transfection, the process is quick and the results also do not last forever, because we add simply add plasmids to the cells that we wish to see the replication in, whereas in a transduction, the results are permanent because it takes much longer, and also because we must add virus to "infect" the cells with the plasmids so that it will permanently change the DNA of the cells and their future lines. to start this process, the first thing we needed to do was a mini prep, which is basically the process in which we isolate the plasmids (DNA) from the bacteria. unfortunately, something got in the way this day, and it was more important that some cells get replated, so instead of doing all of this, we talked about it, and I replated a set of lung cancer cells that had become too confluent.