Hungry Cells on Thanksgiving

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Wednesday marked the start of IL3 withdrawal assay, take 2! This time, I used two different concentrations of fetal bovine serum (10% and 6%) in the media.
One batch 10% serum, one batch 6% serum.

I spent the afternoon at the lab in the hood:
After taking the 7 cell lines out of the incubator, I counted the cells using the Guava Viacount software (I looked it up, it uses flow cytometry--figure below). Basically, the sample is mixed in with Guava "juice" with a 1:10 ratio (for a total volume of 200µl, so 180µl of Guava juice and 20µl of cells), which stains the cells for the counting process. The number of viable cells/mL is the important figure to record.


www.abcam.com/ps/CMS/Images/Flow-Cytometry-how%20it%20works-Diagram.jpg



For an IL3 withdrawal assay, 2 x 10^6 cells are needed from each cell line, so with some simple calculation, I solved for how many mLs I needed from each flask. Since I needed cells for two media compositions, I calculated 4 millilon cells. 
Next, I transferred the appropriate amounts into 15 mL tubes and spun them down in an ultracentrifuge to pellet the cells. Removing the media with an aspirator, I then proceeded to wash the cells 3 times with non-WEHI (IL3) treated media (with 10% serum). This process washes out remnants of growth factors. The washing involves resuspending the pelleted cells in this media, spinning them down, removing supernatant, and repeating. Before the last spin, I split each of the cell lines in two. 

Finally, after the last wash, for the first batch of cells, I used 10% FBS media to resuspend the cells and for the second batch, 6% FBS media.
I moved the cells to clean, new T25 flasks, where they will be living for the next week and a half or so. My mentor plans on counting on Saturday, and then after that, I will be counting the cells every day from Monday. Hopefully, we'll be able to get a better curve than last time (a negative exponential curve (if cells naturally die without IL3) or a positive exponential curve (if cells proliferate)).

Comments

Guava juice sounds delicious!

What was sub-optimal about the last growth curve you generated from your cells?

Future experimental plans: Have you gotten to talk with your mentor and come up with a list of what experiments you'd like to do on this project and when you'd like to schedule them so that you've got all the data you need by the beginning of March?