Properly designing a drug treatment assay measuring cell cycle arrest - (Aug/27/2012 )

Hello,

I am attempting to measure cell cycle arrest in multiple cell lines when treated with a single drug. I have already determined the range of concentrations over which the drug is active, but am still wondering how best I can standardize the experiment and if you have any suggestions regarding how I should be designing my experiment. In the past I have plated the cells at a little less than 50% confluency, waited ~24hrs (at which point they have reached about 50% confluency) and then drug treated. Should I be doing this any differently, i.e., should I be drug treating at the time I seed the cell, should I wait 6-8 hours after seeding, should I base the point I drug treat off of the plates confluency (e.g., drug treat when plate is 50% confluent)? Additionally, should I drug treat the cells in serum free media so that they all synchronize their cell cycles?

Essentially my questions are as follow:

1) When should I begin to drug treat after seeding my cells
2) Should I be treating the cells in serum free media so that they are all in the same stage of the cell cycle upon drug treatment?

Thank you very much

The answer is:
depends on what is the drug doing to your cells and what you want to prove.

1) I would always add the drug after the cells are happily attached; depending on the cell-line, this can be 6 hours after or the next day. With the confluency, it sounds good to me what you are doing now. The only thing I may add is that depending which pathway you are working with, it might be the case that difference confluency leads to different results. For example, a few years ago, when I was working on the sphingosine kinase (SK) pathway and I was looking for drugs that inhibit SK, I observed that the enzyme is anyhow inhibited on its own (in control plates) after 3-4 days due to raise in confluence. I am sure that SK is not the only one doing so. But this is why we do controls and we always compare with controls. Moreover, you might consider the initial confluence based on the timeline of your experiment and the growth rate of your cells. If you need to measure the proliferation over 4 days and you are working with cancer cells, 50% is okish, even though 30-40%ish would be better. (again not all the cell lines are the same) More, depends on the timeline of your experiment.
2) Depends what you want to see; if you are watching for a certain component of the cell cycle, it would be advisable to synchronize them because otherwise your effect will be leveled out by the fact that not all the cells are in the same stage. On the other hand, if it is not crucial for them to be in the same cycle stage, you might think in terms of: when we try to cure cancer in a person by chemotherapy, the cancer is kind of a ball of cells not all synchronized, so better develop drugs that work on non-synchronized cells

Andreea

I agree with ascacioc. If you are watching a specific phase in the cell cycle, then you should probably try to synchronize your cells. Alternatively, if you are conducting the study with an eye towards effects on the entire cell cycle, you might discover that the drug selectively inhibits certain steps (e.g. S phase to G2 phase transition). If you synchronized your cells, it's possible that the effects of the drug would also inhibit an earlier step, and you might not observe the effects on subsequent steps of the cycle.