drewgehring

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