Sorry there has not been an update this week so far, it has been really slow/really busy. Slow in that the only thing I have been doing in the lab is switch the media in the infected cells to the high dosage of antibiotic and maintain it, busy in that I got a job at the theater and have not gotten home until late for the past few days (including today, actually).

The cells worried me for a bit because it did not look like any of them were dying. They had actually looked like they were growing too fast, but I continued the dosage as instructed and they pretty much all died within a 36 hour period. They now look normal with the mock cells at the lowest density, followed by the .1 MOI cells and then the 2.5 MOI cells have the most amount of cells still alive. I have to continue the high dose at a constant rate until all of the mock cells are dead to make sure that all of the cells that we stain are infected.

Just a quick recap of things present and soon to come: The media that the infected cells were in originally is what we are going to use to count the exosomes to determine the cell lines that produce the most and least exosomes, but we need to stain the cells themselves in order to show that we did the infection properly for both  antibiotic resistance and PLAP over-expression. Once we have confirmed the cells were properly infected (probably within the next week or so), we can count the exosomes in the media.

I wish I had something else to tell you, but there really hasn't been anything going on this week. I might be on for a paid summer internship continuing this project, but it has not been finalized yet.

So yesterday and today I began the process of killing all of the cells that weren't infected with virus. It involved math and significant figures. I calculated the proper volume of antibiotic necessary for the low dose (on Saturday) and the high dose (today) when given the total volume of media I needed, the concentration of the antibiotic, and the needed dosage (in micrograms per milliliter). After the math, I actually had to make the antibiotic-media, which was pretty simple, I just put the proper amount of antibiotic into a 3.6 mL aliquot of media. Then I performed a basic media change on the cells, switching the normal media with the antibiotic one.

In other news I saw those stained slides of rat liver and heart and they were very pretty. I wish I had pictures, but I had left my image-taking-device upstairs. Also the liver had a lot more fibrosis than the heart. It was like a swirling vortex of red, purple, and blue whereas the heart was almost entirely red.

Tomorrow I'm going to switch the cells to the high dose and then keep them at that constant level until all of the cells in the wells with no infected cells die. I don't know how long it will take, probably a week, but then we can freeze some of them back and stain them and actually show that we infected everything properly and get a preliminary report on how much PLAP they are overproducing.

My HeLa cells are growing slowly. Very slowly. But that's okay, because they've reached the point where they've been passaged so many times that it kind of wears them out. Also, we stained the cells that we mummified earlier and it totally worked! But we forgot to freeze some of the virus-infected cells before we mummified them, so now we have to do it again. But this time I get to make the antibiotic media and dose the cells myself.

Anyways, when we stained the cells we had to rinse them in a bunch of stuff and then wrap them in foil. So they went from this:

To little miniature Ding Dongs:

Sure, they look adorable, but the sound of latex on aluminum is NOT fun. But the stain worked, and the some of the pictures that came out of it looked like they should be in a brochure trying to convince kids to go into science, so I'll try to get some of those pictures to put up when we stain this next batch.

I'll have to go in on the weekends in order to continue upping the dosage of the antibiotic. And I have to do math to figure out the proper volume of antibiotic to put in each well. Math with unit multipliers. Not going to lie, it's pretty intense.

So, I had to make more media today, since I have to keep growing cells because they suddenly decided that the middle of the flask was overrated. Also we have to thaw some more Neuro 2a and SY5Y cells because they basically did the same thing expect they decided the whole staying alive thing was just too much effort.

Yesterday we mummified PC12, HeLa, and H4 mock cells to measure PLAP levels against those that were infected. But we had to use the regular cells because all the mock cells that we had earlier in the infection process were exposed to the antibiotic. So they died. We also took the media from the infected PC12 cells for the RNA, DNA, and protein using the process that I now have the protocol for. This protocol comes with a kit with all the necessary parts.
So, according to the diagram in the protocol that won't paste into this thing, the cells get lysed in this tube, then put into a "Allprep column" and centrifuged so the DNA stays in the upper portion and the RNA and protein flows through. To purify the DNA, the top portion is washed and eluted (which means to remove an adsorbed substance by washing with a solvent). Then ethanol is added to the bottom part with the RNA and protein. Then it is placed in a "RNeasy column" and centrifuged to a point where the RNA remains in the top and the protein falls through. The part with the RNA is then washed and eluted and the bottom part with the precipitated protein is centrifuged and redissolved.

We're going to have to prove that the cells were actually infected as planned, so we have put some of the infected cells and essentially mummified them so we can see how much PLAP protein they are producing. We had to wash them with PBS, then soak them in 4% PFA (paraformaldehyde) and PBS, then keep wash and leave them in PBS so they don't dry out. This process 1) gets rid of everything that aren't living cells and 2) preserves the cells in whatever state they were in. We have yet to stain them and measure the PLAP.

In other news, I watched a staining of those kidney and heart tissues I saw on Saturday. Side note: The tissues were from rats. Anyway, the process was very time-consuming and precise. There was much soaking in many  colorful fluids for differing and specific amounts of time. Most of which smelled terrible and would hurt your eyes if you looked at it too long. Like Xylene. Xylene sucks. But the end result was this:

Today we started a process that will take the exosomes from the media, and then separate the DNA, RNA, and protein from inside the cells. I wasn't there for the media bit, but I was there for the separating the cells from their innards bit. So we took the cells that produced the media we harvested and resuspended them in a lysis buffer which turned them into gross semi-opaque bubble-goo. We put the mess into these tubes that were a part of a kit to purify RNA and DNA and put them in a centrifuge. The larger DNA and protein would stay above the filter whil the RNA and other cell guts would slip through to the bottom. Then I had to leave, but Mari is sending me the protocol so I could sum this up better.

Sorry this post is a little late, I was very busy yesterday preparing for Easter today, but thankfully I've gotten a bit of a break today, so, I'm writing the post now. Unfortunately, we weren't able to do anything else in the lab, because I was unable to come in on Friday, and nothing happened on Thursday because even though I thought things were going to happen, one of my lab coworkers (I guess that's the right word?) didn't show up, so we couldn't get anything finished. On Saturday, as I said I was tied up, but I managed to nip in just long enough to perform the fastest cell split ever and look at some slides of heart and liver tissue before heading back home.

So, to sum up, we have, last time I checked, two cells lines infected with virus and antibiotics, and the rest have yet to be transferred over to the wells. Should be getting more stuff done tomorrow, people most definitely going to be there. I'm going to try to get the protocol for refining the RNA, DNA, and protein from Mari because even though it's not technically part of the project, it's still interesting and a wide spread procedure. So, hopefully by Monday we will be able to get some more stuff done to get the cells on their way to being killed for science!

So I Just Learned Something New...

Those cells that I have been splitting and taking care of are apparently one of the most famous cell lines ever. They are called HeLa cells, which stands for Henrietta Lacks, an African American woman whose cells were taken for research without her permission. Every lab that uses HeLa cells are all using cells whose ancestry dates back to this woman. The cells have been in use for more than 60 years, which is almost twice the amount of time Henrietta was even alive. It was very controversial because both the cancerous and normal cells were taken without her knowledge or consent. However, these cells were particularly helpful for research because of their ability to survive after a few cell divisions. This allowed the scientists to perform more experiments rather than focusing on keeping the cells alive. Her cells have been used to research everything from the cure to polio to potential treatments for AIDS to potential sensitivity to miscellaneous products like make-up or glue.

Yesterday I watched a bit of a procedure that purified RNA, DNA, and protein from brain tissue. It involved a lot of washing via copious centrifuging. The wash would drag the smaller particles through a filter during the centrifuge, leaving the RNA in the top portion of the container. The DNA was in the process of being separated from each other in a different centrifuge.

Hopefully we will be able to count the PLAP protein from our cells by the end of the week, but I really don't know.