So, yesterday we were able to infect the two cell lines from earlier in the week. It didn't take much, actually, we just emptied the wells of media, rinsed them, then put new media in which had a calculated amount of virus int them. We have to put the virus into the media before we put the media into the wells because it helps spread the virus more evenly over the well surface. If we put the media in first, the virus would be concentrated in what ever section we put it in. This way we maximize the number of cells infected with the virus to more closely approximate the number that we calculated.

Also, random note, everything that touches anything that might have had contact with the virus goes into bleach. Which we then let soak. Overnight. With the UV light on. It's not harmful, but we're not supposed to take chances. It might get into some other things that aren't supposed to have PLAP protein-making virus in them. 

Today I split my cells. They're still alive. But since today was a Saturday, no one else was in the lab. Sometimes people will come in to work on the weekends, but today there was absolutely no one. It was kind of awesome.

Hey Look! Another Update!

So, I am being tested on my ability to conduct procedures in a fume hood correctly, efficiently, and sterile-ly. To this end, I have been delegated a single flask of HELA cells to grow for a week in media with antibiotic in the media, then I have to grow them further without the antibiotic to see if I'm not messing up. Good news. I'm not messing up. I have been caring for these cells for quite a while now, and they are decidedly not dead. Just changed their media today, and they are showing a startling lack of contamination.

Also, we managed to actually seed two cell lines in a couple of 6-well plates like we were supposed to, and they should be ready to infect with virus tomorrow. After which I will tell how that went. Anyway, the only thing that really happened between what I told you about last time and finishing seeding the cells was we added more media so we had enough of a volume to out in each well, then we calculated how many mLs of the new solution would give us the proper number of cells per well, then we added them. And labelled them. The labeling was important.

We are going to kill all of the cells not infected by virus, too.
And some wells have more virus per cell, either .1 or .5 MOIs.
I think I already told you that.

But we can't just dump a whole bunch of ampicillin into the wells. That would kill the cells that even had the immunity from the virus. We have to acclimate the cells to the ampicillin's presence. Then, once the ampicillin has won the trust of the viral-cells, we will rampu up the dosage, killing all of the cells not infected.

Random Stuff That Can't Go In It's Own Blog Post Because It's Too Short

When we use the virus that we aliquot, we have to refreeze it every time, but since refreezing damages the integrity of the virus, we have to use each aliquot before going back to the first one. So if the aliquots were this string of numbers were each number represented the number of times it had been used, it would look like this:
0000
1000
1100
1110
1111
2111
2211
etc.

The difference between transfecting and infecting and transforming is that infecting means that you are infecting cells with a virus or bacteriophage, transfection is when you introduce DNA and RNA into cells by other methods like chemical introduction, and transforming is the same thing as transfection except with bacteria.

I do other stuff during my time at the lab that isn't strictly related to my project, just so I can see certain procedures that we will use later or to gain more knowledge in general (like making cDNA from RNA or re-organizing the freezer).

I also had to take a course on ethics and proper laboratory practices when applying for a grant or writing a paper or using human test subjects. It was really long and legally mandatory.

We will eventually have 5 6-well plates each with three wells filled with a particular cell line. One well will be infected with .1 MOI (unit of infection essentially one virus per ten cells), one with .5 MOI, and one with no virus at all. The virus serves a double purpose of over expressing PLAP and providing the infected cells with a resistance to a particular antibody. The last one will be used as a control in order to determine the amount of time it should take to kill all of the cells NOT infected with the virus so we know when the cells are done dying in the wells that DO have virus in them.

Titurate: to pipette up and down in order to homogenize (mix) a solution.

Today I watched the beginnings of a TaqMan Gene Expression Assay, which is basically turning RNA into cDNA, then attaching probes and quenchers to the cDNA, then putting it into this machine which fluctuates the temperature which somehow amplifies the signal that the probes are giving off so that when the probe is measured we can pick a minimum number of times we want a certain sequence to appear, and then we can determine which samples have how much of that sequence compared to each other.

Since we are going to be using viruses a lot in the next week or so, I think I should probably tell the Internet more about viruses, both viruses in general and in the one we are using in particular. I wasn't a part of the virus-making process, but I was part of the plasmid-isolating-from-bacteria-to-put-in-the-virus bit. We used a MIDI prep in order to isolate the bit of DNA we wanted from the bacteria it came from. Bacteria smells absolutely terrible, by the way, not in an overtly bad smell, but like a normal smell that is just, off. Wikipedia has a pretty good article on MIDI preps here: http://en.wikipedia.org/wiki/Plasmid_preparation. The bit of plasmid in question happened to have the magical ability to over express PLAP, the marker that we were going to use to count exosomes. By isolating it using this MIDI prep, we can then put it into the virus which will the infect the cells and tell them to produce PLAP and package it in exosomes. Next in the timeline, the virus was actually made and aliquoted, split into a whole bunch of equally sized volumes from a large volume.

This week, we have to first seed the cells onto a six-well plate with a certain number of cells in each well. It starts like a regular cell split, but after deactivating the Trypsin, it actually has to be centrifuged to get rid of it altogether because we don't want it interacting with the stuff we use to infect the cells with virus. Then we put regular cell food in there, then, after mixing the cells back into the media, we take a bit of the mixture, put it onto a special plate, then stick it in- I kid you not- a cellometer. Which counts cells. Then we do some quick math magic and Abracadalgebra! We have the proper amount of media to put into each plate to make sure enough cells get on there. I don't have anything after this because when we tried to seed the cells last time, science failed and with one kind of cells, there weren't enough in order to have the proper amount in each well, and we dropped the conical containing a different line of cells, so we have to wait before the other flask is ready to be seeded, especially after we split it into two flasks so we have another back-up.

I think that it would be beneficial to go over the previous research as it pertains to this project. The most influential paper can be found here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0031302
However, that paper is very technical and difficult to understand, so I'll sum it up for you now. Basically, Tauopathy is a fancy word for tangles of Tau protein in your brain that cause bad things to happened and brain cells to die. This paper begins by telling us that tauopathy starts in a bit of the brain that's in the middle. The point of this experiment was to determine whether the tauopathy spreads along synaptically connected pathways, which means that their testing to see it the bad stuff spreads through touching other things or if it's caused by something else. They used a "transgenic mouse model" in order to track the tauopathy through the mouse brain throughout the course of their lives to see if the different brain pictures matched those of an equivalent human brain. By measuring the progress of the disease in mice brains, they could extrapolate the same path the disease would take in human brains. They wanted to know whether the tau was transferred in between synapses or if it developed independently. If the disease was transferred via synapses, then it was possible that the Tau might be contained to a certain region of the brain, not being allowed to spread farther and cause further damage to the brain. They tracked the progression of the tau by detecting it with MC1 (monoclonal antibody) in mouse brains ranging in age from 10 months to 22 months. They came to the conclusion that tau did spread trans-synaptically and could possibly contained to a specific part of the brain to prevent further damage.

We are just about ready to infect the cells with virus that will enable us to count the number of exosomes. The virus we have delivers both the marker and a resistance to an particular antibody, so once we let the virus settle, we can treat the cells with the antibody, and all of the cells not infected with the virus will die. This way, the only exosomes we will count will be produced by the cells infected with the virus. We changed our plan a little though, and instead of choosing the cell lines before infecting them, we are going to infect them first and choose the best cells lines after counting the exosomes produced. This way, we can bypass the necessity of exosome-free media.

On a different note, I think I should explain what exosomes actually are. They were discovered relatively recently, and were thought to be just little garbage cans of the cells. But in 1996, they were discovered to also function as a type of intercellular communication. They could be packaged with anything from proteins to digested pathogens to bits of RNA, all of which can get transferred and integrated with other cells. They ar involved in maintaining the immune system, although they can cause both beneficial and harmful effects. They are also have the same relationship with cancer, sometimes hindering and sometimes helping its progression. They often have receptors on their membrane that correspond to different types of cells that they communicate with. Occasionally, they will carry in them the necessary component to trigger apoptosis (cell death) in the receiving cell.

Update

Well, for the past week, we haven't been doing anything intensive. We have mostly just come in, checked the cells for growth then split them or changed the media if necessary. Today we put one of the cell lines into 6-well plates, and that was a different procedure because instead of measuring using volume, we had to get a specific number of cells into the well regardless of concentration. We had to use a cellometer to measure how many cells were in the solution, then we had to dilute it to the correct volume for the total volume that was going into the wells, then we actually put it in the wells.

I have to check on the cells everyday, and now we have to make special media to prevent the exosome count from going haywire because of the exosomes in the FBS that we have to put in the media. So we have to put the media in a special centrifuge that takes 16 hours to complete. So this part takes a lot of patience and timing.