I went to the lab on Monday to begin a new IL3 withdrawal assay with 6% serum. This assay includes all four mutations from the four different cancers (3 in NTRK1 and 1 in NTRK2), as well as NTRK1 and NTKR2 WT, and the control, pMSCVpuro (retroviral vector w/no NTRK). Just for more info: pMSCVpuro is a retroviral vector (plasmid) "optimized for expression of a gene in hematopoietic, embryonic stem, or embryonic carcinoma cells" (AddGene).
So I began the process of the IL3 withdrawal by counting the Ba/F3 cells that were thriving well and healthy in IL3 containing media. That way, I could calculate the same amount of cells I was going to spin down from each cell line. The numbers that are crucial to note are the # of viable cells per mL and the % viability of the cells (Theses numbers also count as Day 0 counts). Next, I washed the cells three times (spinning, washing with fresh media w/o IL3) to get rid of any growth factors left in the previous media that might affect the growth of the cells in the withdrawal.
Media I used to wash: to make 6% serum, I diluted the 10% FBS media down with RPMI-only media. Ex. 6mL 10% media with 4mL no FBS media.
With the washing finished, I transferred the new cell cultures with 6% media to seven T25 flasks, labeled, and incubated.