On Friday, I went to the lab to continue my cell counts for the IL3 withdrawal assay in 6% serum (Day 10).
After counting, I split the NTRK1 WT cells, since the media became quite yellow.
Next, I prepared to serum starve the batch of NTRK1 and NTRK2 Ba/F3 cell lines that were recently thawed out and growing in media with IL3.
Purpose: to serum starve the cells, then lyse those cells, and repeat western blots. In the previous phosphorylation western blots, the cells had been growing in media with serum. In the serum, there are a lot of growth factors. These growth factors may prompt phosphorylation (for example, last time, the control expressed MEK which should only be expressed in the NTRKs), so to get rid of that influence, I need to starve the cells of serum, so there are no external factors that may be causing the phosphorylation.
I counted and calculated the same number of cells to spin down for each cell line. After spinning, I removed the media, washed once with RPMI only (no FBS, no IL3), spun down the cells again, and sucked off the media. I resuspended the cells in 5mLs RPMI only media in T25 flasks for an overnight starvation.