Well, we still don't have any results, nor have we begun the staining/freezing process because unlike last time, we need to grow a bunch more cells in order to have enough to freeze back. So we needed to grow them from the little twelve wells into the large flasks that we were using in the beginning. But we can't just transfer immediately into them, because there aren't enough to stimulate growth.We have to switch them from the small wells to a larger well to a small flask to the large flasks. The high infection cells are being switched to the large flasks tomorrow because they have a higher cell density the the low infection, only one of which has even been able to be transferred into the larger well. In conclusion, I'm going to continue going into TGen even though the scheduled internship is technically over so I can try to get some final results before the presentation is due. I also have been accepted as an Ivy Scholar which will give me a paid internship at TGen during the summer, so it's possible that I could finish the entire project and not just this portion. Maybe. I'll probably stop updating this though, because I have a bunch of other things I have to do and I'm pretty sure that internship is 40 hours a week.

Hey, so this week has been really slow as well. I've just been going in, switching the media for the infected cells and maintaining my HeLa cells, so I've only been going in for about an hour a day. Fortunately, the mock cells in the infected plate have all died out, which means that we're pretty much ready to freeze them back and stain them, which could happen as soon as this weekend. But I'm not sure, so this week might be a little short...

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.

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.

This was a busy week

So much stuff happened this week, that writing down the protocols for everything and explaining what they mean would take up enough space so I wouldn't have to do my blogs for the rest of the project (If I could write it all at one time). It started on Wednesday with day one of two of the western blot (a procedure to test if a specific protein was in the sample). This particular western blot was not part of my project, but since we would be doing one later, we figured it would be informative if I knew the process beforehand. It involved making samples with a lysate and water, putting it between heat blocks (exactly what they sound like) and then into wells in one of these:

along with ladders (not actual ladders), filled with buffer solution, then packed in ice an left to sit for 1.5 hours while running at 125V . After that was over, we had to make a "sandwich" of sponges, the gel that was in the contraption, filters and membranes. Also everything was soaked in buffer. Then the sandwich goes back into the western box which is again filled with buffer and packed in ice and left to sit for 3 hours on 125V. Then the membrane with the stuff on it is taken out and poured with Ponceau stain to check for protein. That's when I had to leave for the AMC Math competition, but the protocol for the rest of the day says that it then needs to have a 1 hour blocking with 5% milk (5g dry milk + 100mL TBST) followed by: Incubate in primary antibody (find dilution in AB datasheet, dilute in 2.5% milk) overnight in the fridge.

While that long three hour break was going on, we split the cells from our project. We basically took out the cell food, used Trypsin to break the cells from the wall of the flask, incubated it at 37 degrees Celcius for 5 minutes, then added cell food back into the flask, swished it around a bit, put it in a 50mL conical, broke up clumps of cells using a seralogical pipette, put in media into each of the new flasks, then 1mL of the cell-Trypsin-media went into the flasks.

And that was Wednesday.

On the Past Few Days

On Thursday we concocted media (cell food) to put the cells in. Each line of cells required a different recipe of media, and the entire day was taken up by the project. Most of the media contained a base formula and a kind of serum either FBS (Fetal Bovine Serum) or Horse Serum. Combining the ingredients was a tedious process, and much ethanol was sprayed on gloves, packaged filters, and media containers in order to maintain sterility.

Friday was dedicated to thawing the cell lines that we needed and placing them in a preliminary media to be changed out the next day.

On Monday we checked on the cells to see if they were growing properly, and the PC-12 cells look like they're about ready to be split. Tomorrow will be short, but I will spend the majority of Wednesday and Thursday observing a western blot that we will eventually have to perform on our own cells.

Adios.

Previous Research

This is the research that the lab I working on at TGen is based on.
I didn't think that it was worth setting up a whole page just for one link, so I'm putting it here.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0031302

Read it if you want, but it's a bit technical.

First Day At TGen

As the title says, today was my first day at TGen! Today I found out what I was going to be doing for the next two and a half months and met the people I would be working with.

The brains of patients with Alzheimer's disease are characterized by the presence of Amyloid B plaques and tangles of Tau protein that spread from entorhinal cortex through the hippocampus and to connected areas of the cerebral cortex. The Tau proteins can be transmitted between neurons either through neurotransmitters or through exosomes. Because the cell strictly regulates the neurotransmitters, our lab will be focusing on the exosomes and trying to prevent their spread through the brain. If we can contain the Tau protein, the portions of the brain involving the memory loss typical of Alzheimer's patients could be diminished.

I will be helping Ms. Turk and Ms. Krate carry out the experiment designed by Dr. Huentelman that will attempt to find genetic and chemical inhibitors of exosome production. First, we will take six different kinds of cells (Cath.a, Neuro-2a, PC-12, SH-SY5Y, H4, and HELA) and grow them in a cell culture until they are at 90% confluency (a measure of cell density on the bottom of the dish) at which point we will split them, one to return to the freezer, one to use as a sample. We will then force the exosomes to package a protein called PLAP to use as a marker. After 48 hours, we will centrifuge the samples and collect the supernatant containing the exosomes. Then we will lyse the cells, color the PLAP proteins and keep two of the cell lines based on exosome count. The two chosen cells will then be run through a drug screen and a gene screen to determine which chemicals and genes control exosome production.

I don't have a very regular schedule, so updates will be very uneven. Bye!

 P.S. What is the protocol for signing off of a blog post? Every time I do it, it seems very awkward.

Official Senior Project Blog Starting Now!

Hello! My name is Erika, and I am a senior student at BASIS Scottsdale. During the final trimester the seniors are encouraged to take on a senior project, blog about it on a biweekly basis, and present on the results at the end of the school year. This is my blog. I will be participating in one of Dr. Huentelman's labs at the Translational Genomics Research Institute, or TGen. Dr. Huentelman has a few labs, including ones specializing in Alzheimer's,   and autism, and cognitive aging. The purpose of these labs are to find the genetics factors for the unfavorable trait, and apply those findings to produce a medication to combat it. For example, having the ApoE4 gene in both copies of someone's genetic code greatly increases the risk of late-onset Alzheimer's disease. Dr. Huentelman's lab hopes to find a common section of code that possibly neutralizes the effect of the ApoE4 allele by analyzing the DNA of people who have two copies of ApoE4, but do not have Alzheimer's. One of his labs also focuses on analyzing dog DNA and extrapolating the results to humans. Because of the founder effect, certain dogs are genetically predispositioned for certain diseases and conditions. By finding dogs that don't exhibit the particular trait, the lab can try to pinpoint the section of DNA keeping the dog healthy, and apply that knowledge when looking at human samples. I soon hope to know which lab I will be working, and once I do, I can produce a formal proposal and put it on a page in this blog. Until then, I wait anxiously checking my email.