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.