Juggling

Hello and welcome back!

This week I learned to juggle… that is, I learned to juggle multiple new experiments and strange new jobs.

To start off, though it may seem a little unrelated, I’ve begun working at McDonald’s. It’s been interesting to say the least. Luckily, it hasn’t affected my schedule at Mayo Clinic, although it has altered my outlook a little bit. I think I can appreciate the slow, relaxed, meticulous pace of the lab more now that I can compare it to swearing customers, burning French fries, and toppling ice cream cones. However, as different as it may seem, I’ve been able to draw some parallels between the two; like the fact that in either work place I’m constantly sanitizing. In one job I’m growing bacteria, and in another I’m getting rid of it…and I honestly couldn’t tell you which one is which.

But, regarding juggling experiments at the Mayo Clinic, I’ve now got 3 active experiments going at the same time. One experiment is the drug titration, which determines the progress of cell proliferation. Another is the analysis of the RNA produced by each cell line. And the last is the western blot, to determine the protein production. All three steps will be the final determination of what our conclusion to the experiment will be. The end is approaching, and it’s exciting stuff.

The results from last week’s drug titration also came in, and it seems that there may be some off target effects coming from the inactive version of the drug. The inactive versions are meant to act as a control, having the same effect on the cells as those that have not been treated. However, when we reviewed the results, in some cases the cells treated with the inactive drug grew even more than those with no drug. This may hint that the drugs are having some unwanted side effects. But, we’ve decided to run the test again to make sure that our results are more certain.

Until then, thank you for reading!

Angela

Experiments: the endless cycle

Hello and welcome back!

The results are in! And they’re consistent—something I hadn’t realized was so important to experiments until now. After putting the U266 (the cell line that reacted to the drug, even though we didn’t expect it to) through a drug titration (an experiment to see at what concentration the cells are affected) it showed marked signs of inhibited growth when a drug concentration of 1 uM was added. This is good news, since it not only verifies that we are following the drug company’s protocol but also that I am not horrible at lab experiments.

Unfortunately for me, I must’ve done too well—because this week I had to extend the drug titration to all four cell lines. That means 16 different drug concentrations, 160 wells, and 800,000 cells. My poor thumbs.

We’ve also begun to look at the protein and RNA synthesis of the treated cell lines. The ASO’s are meant to decrease production of IRF4 by inhibiting its RNA production. So, we should expect to see a marked decrease in RNA and protein production in cell lines treated with the active drugs. It will also be interesting to see the results of the cell lines that did not react to the drug. It may give us a clearer insight as to why the drug has no effect. We will be using a western blot to determine protein production (something I’ve heard about in Biology class but have never tried). It’s bound to be interesting!

Until then, thank you for reading!

Angela

A solution

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

Killing it

 

Hello and welcome back!

Firstly, I’d like to calmly inform you that…

THE EXPERIMENT WAS A SUCCESS!!!!!!!!!!!!!!! THE CELLS ARE REALLY REALLY DEAD BUT IN A GREAT WAY!!!!!! LOOK AT MY 12 WELL PLATE WITH ALL THE DEAD CELLS IN IT!!!!!!!!!!!

 

 

Ahem.

So, from this experiment we can conclude that the drugs are, in fact, working, which is a good start. This was, however, only tested in one cell line.

This week, I began applying the same experimental conditions to other cell lines with different genetic variables. One of these cell lines (the one with the results above) is especially sensitive to IRF4, while another is not. So, the next experiment will hopefully show the drug’s effect on the one cell line but not on the other. More cell lines are being tested and if the hypothesis hold true in this next experiment, we may be able to answer more questions about those cell lines.

 

So, in preparation for the next experiment, I’ve plated 48 wells of cells via pipette (I’m not sure if you know how much thumb work that requires, but let me just say that challenging me to a thumb war should not be taken lightly) with the placebo and drug. We will be testing the effect of the drug by counting the live cells, for now. Later, we hope to test the mRNA and protein production. We may even throw in a titration of the drug. Who knows? Endless possibilities when it comes to killing myeloma cells.

 

However, there is some bad news. Now, two lines of my cells are dying. It seems there may be a pattern here… I’ve replaced them with fresh cell lines, and hopefully they will live to die in the next experiment.

Regardless, thank you for reading.

Until next week,

Angela