Lauren Donato Week 2 B Cell Growth and Data Analyzation

Week two at the Mason Lab has been just as great as the first. I am learning skills that are going to be useful throughout the rest of my life. I went to another lab meeting on Tuesday and presented to the other lab members what I did the week before. There was donuts, Dr. P was right about the donut thing. 

Dr. Mason at our lab meeting  & Donuts ^^

Now that I have proved that I am more than capable of working here at the Mason Lab, Dr. Mason and others in the lab are assigning me more essential tasks without much supervision. It is pretty challenging at times, but I am becoming increasingly confident in my skills here at the lab, and feel a sense of expectation and trust. Dr. Mason continues to make me laugh all day,  as we talk about England losing the World Cup and ways to help my fat shih tzu Tom. 

fat tom ^

This week I continued my B cell work, and also aided other projects coming to a close in the lab. The growth curve of B cell appears similar to the curve of the productivity of an enzyme before it becomes saturated with substrate. The B cells proliferate until they level off. When they level off, it is time to restimulate them. I have been stimulating the B cells we have grown last week with cytokine IL4 and cyclosporine (which prevents T cells from expressing). I have also been adding fresh B cell media to each well, and K562 cells. Making B cell media is a task you have to be very careful doing, for contamination has to be avoided. It is similar to cooking, as in you get a "recipe" and add the correct portions of each, until the "flavor" or color of there media is just right. Some ingredients in B cell media include: gentamicin, glutamine, human transferring replacement, human AB serum, HEPES buffer, and Iscoves MDM. While making B cell media, I work in a tissue culture room under a hood and keep everything clean and sterile.

what b cell media looks like

 To keep track of B cell growth, I suspend the cells and count them under a microscope. This involves diluting the cells, staining them, and then using a hemocytometer or "counting square" to count the cells. I have become a pro at this, while on day 1 it was very intimidating. I have been keeping track of my B cell growth on an excel spread sheet by making graphs that display different kinds of data. 

hemocytometer

Aside from my B cells, whenever Dr. Mason receives blood from a patient in the morning, I do a PBMC of the blood and freeze down the lymphocytes. This gets harder when she gives me 4 patients blood to do at the same time, haha. During a PBMC, I dilute blood, ficol, and PBS in a 1:1:1 ratio. I then layer the blood and pbs mixture on top of the ficol, careful not to disrupt the clean layer, and I centrifuge it. 

layering of diluted blood and ficol

When out of the centrifuge the lymphocyte layer is apparent in the middle of the tube and I pipette them out and count them.

diagram of PBMC isolation post centrifuge

 Once counted, I freeze down these cells by using freezing medium. We call the freezing container "Mr.Freezey". I transfer the cells to a -80 degree Celsius fridge, and then the next day to a liquid nitrogen tank that goes as low as -180 degree Celsius.

liquid nitrogen tank

 My experience with this the first time was scary! Lots of steam comes out of this tank and I have to be careful it doesn't leak. This week I also I became familiar with a neutrophil assay and flocytometry. The neutrophil assay was a long protocol or a "lenti" process, but very very cool. I cannot use the flow cytometry machine (FACS) because I am not trained, but I watch how it works and understand it. 

FACS machine

While the osteosarcoma project is coming to a close, I have been doing a lot of work for Dr.Mason, constructing graphs of the data using excel. This was pressuring because I have avoided learning excel for a while, but I now am a pro. She used a lot of my graphs in a talk she gave on Thursday. I finally got my Penn ID card on Wednesday, which was exciting. The penn bookstore is really nice and so big.

PennCard Center at the penn bookstore :)

 I have been staying in the lab late this week, but the days go fast. Lab on people !