Hello and welcome back,
The results of the new experiment are in, and are filled
with surprises. After conducting a MTS assay on all four cell lines, we were
able to see the effect of the drug on cell proliferation by measuring the ATP
produced. Though we did not know exactly what to expect, we did have the
hypothesis that the U266 cell line would be resistant to the ASO’s because it
does not produce MYC, and—we thought—would not be dependent on IRF4 because of
the lack of MYC. However, we saw that the ATP production of U266 was cut in
half when administered the active ASO’s. This hints that U266 may still be
dependent on IRF4, despite a lack of MYC production. In contrast, the FR4,
which does produce MYC, was not affected by the ASO’s at all—a very unexpected
turn of events. We will be examining these cell lines in depth in the upcoming
weeks to determine just why this is the case. Interestingly, the KMS11 and the
XG7 reacted as expected.
But, now that we’ve answered the question of which cell
lines are reacting, we’ve decided to test another direction of the experiment:
determining at what concentration of the drug the cells become sensitive. So,
we’ve set up drug titration experiment, whereby we’ve plated one cell line (in
the same reduced concentration of 5K cells/100 ul) with varying concentrations
of all the ASO drugs (active and inactive).
Of course, this sounds easy enough. But. It. Took. Forever. 40
wells, 16 stocks of drugs, and 96 pipette tips later, the plate is set to sit
in the incubator for another 6 days before I can go through another 40 tips and
a tube of cell titer-glo to see the results.
Until then, thank you for reading. Happy pipetting!
Angela
I'm glad your results are starting to come in. Do you use a plate reader to determine whether or not stuff is dead or is it more of a binary thing you can tell just by looking at it?
ReplyDeleteA bit of both, actually. We've been using an MTS assay to measure the ATP production in the cell lines quantitatively. But, qualitatively, you can tell just by the color of the cells whether they're doing well or not.
DeleteAngela....it appears so far that you are well on your way to having a presentation that will actually be able to make a conclusion! So many of the projects, due to time constraints are not able to draw any conclusions. Your posts are all so interesting. It also make me feel good that I can actually follow what you are doing!!!! Keep it up.
ReplyDeleteSo which cell lines are you testing? And are all the cell lines still survivng?
ReplyDelete