This week at the lab, my mentor and I went over the protocol I wrote (which I can show you or email to you) for the serum dilution assay, gathered all of the data from the IL3 withdrawal assays and serum dilution assay, and made graphs in Excel.
For the serum dilution: The bar graph measures average absorbance (y-axis) depending on the concentration of serum used in each well (10% down to 1%). Although the mutant showed more variance, there didn't seem to be much overlap between the wild type cell viability and mutant cell viability. We determined that this round of findings was significant. (relevant to my project, NTRK2 mutant)
What we did yesterday was compare the graphs of readings done after the chemical reagent MTS had incubated (at room temp) in the wells for 2 hours with a reading after 6 hours.
The increase in the bars was linear for both the wild type and mutant--not much changed. Therefore, a reading after 2 hours is fine.
Overall, the mutant cells outgrew the wild type cells.
At 6% serum, there showed a clear difference between WT and mutant cell viability.
Graphs for the IL3 withdrawal assays were a little strange. The mutants that died off eventually had nice looking lines, but the ones that grew spiked and decreased in some places. Cell counts would go from numbers 10^5 with high percentage cell viability but then the next day have counts at some 10^6 with low percentage cell viability--I inputted only the number data, not the percentages
Note to self: carefully mark in lab notebook whenever I split cells (a likely reason for the jumps in numbers), and very clearly mark the dates as well as day # of the assay every time I count cells (not just Day 1, Day 3, but ex. November 14, 2013, Day 8)
1. we have to redo the IL3 withdrawal assays anyways, but with the new information from the serum dilution assay:
redo the IL3 withdrawal assays with all the mutants and wild types, two times (one with 10% FBS and one with 6% serum)
2. finish counting colonies in soft agar
3. also, now that I have some evidence for the NTRK2 mutant I am working with--it's showing signs of transformation, more transforming compared to WT as shown in serum dilution--there is much more I can do to study the pathway of this mutation
-soon, I will try a western blot
À la prochaine!