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.
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)).