Part 1: Answers to Follow-up Questions

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Which mutation/cell line looked the most promising at 6% in the last assay you ran? Have you run a full IL3 withdrawal assay for all 4 lines of cells? Is there a reason to expect that all 4 mutations would behave similarly at 6%?

Thank you Veronica for the great questions. They make me dive deeper into the reasoning behind my research and also prompt me to be more specific when I describe a certain experiment. 

Yes, all the IL3 withdrawal assays I have done included all 4 cell lines. Now in my results section of my research paper are images of the flasks from the last IL3 withdrawal assay I ran, which had both 10% and 6% serum. Unfortunately, there isn't a graph for these data (the cell counts were unfinished), so my comments are based solely on observation. This means I'm looking at the color changes in the media across the 4 cells lines, especially differences between wild type and mutation.

I talked a little about my observations of the flasks here but I will go into more detail below. 

In 10% serum
NTRK1. From what I noticed, there was only one NTRK1 mutation that was transforming (media with yellowish: cells are dividing quickly and using up resources--cell-sitting blog post). NTRK1 wild type was also transforming (similar shade (?) of yellow).*
NTRK2. For NTKR2, not only did the mutation look transforming (yellowish media), but also the NTRK2 WT (dark yellow media)**

In 6% serum
NTRK1. Media was darker yellow for both NTRK1 WT and the same mutation that transformed in 10%. 
NTRK2. Huge difference. NTRK2 WT did not transform at all. The media was a normal red. NTRK2 mutation media was yellowish like in 10% serum. 

Conclusions: NTRK1 WT behavior does not differ much between 10% and 6%. Only one NTRK1 mutation seems to transform in IL3-free environment. The difference between NTRK2 WT and NTRK2 mutation behavior shows when serum is diluted down to 6%. I'm thinking about how to approach the investigation/analysis of the how and why there is "something" that occurs and causes this effect.

The current IL3 withdrawal assay I am working on only tests the cell lines in 6% serum, and acts as a proper repitition of the assay described above. So, from my observations from the last assay, I can expect that two of the three NTRK1 mutations will most likely be negative and not transform, while NTRK1 WT and the one other NTRK1 mutant will transform and have similar cell viabilities. In addition, NTRK2 WT should not show any signs of cell survival, and NTRK2 mutation will be transforming. 

If I have time after this assay is finished, I would like to do the assay again in 10% as a control for comparison. 

*I was wondering what this could potentially mean, if the wild type seems to be transforming. 
**Similar thoughts as mentioned above. I have the western blot that shows darker, thicker bands for NTRK2 WT (which means the protein is being expressed at higher levels), but then in 6%, NTRK2 WT does not transform. 

Thanks again for your questions and comments!


fantastic replies!

Wow, these descriptions are specific and clear and very useful - well done!
Flask pics sound great, and it's ok that you have descriptions but not quantification of your results. Reporting details such as media pH and thus cell metabolic rate, or thickness of Western blot band and thus amount of protein, are both valid ways of describing what you're observing.