Drug Treatment and Cell cycle analysis - (Jan/12/2007 )
Hi, I'm a newbie in this area and would appreciate some suggestions.
I'm about to work on cell cycle analysis using PI and apoptotic studies (Annexin V+ PI)
I would be testing out different doses of drug, 5 FU, on a number of colorectal cell lines (colo320 and HCt116 for starters before moving on to further work with transfection of my own gene)
May I know what to get about to know the range of drug concentrations to use and the duration of drug treatments?
I do have a range in mind from reading papers but I just wondered why had the authors chosen such concentrations and duration.
Has it got to do with the doubling times of the cell lines?
When they say a treatment of 72hrs, for example, does that mean treating it on say 0 and harvesting 72h later? Or would we have to change with fresh media (with 5FU) everyday?
Another separate question, I keep hearing people talk about synchronising cells, may I know what's the purpose of this?
In my cell cycle analysis experiments, once I treat the cells I leave them alone until the time of harvest. Disturbing the cells by changing/adding media can affect their progression through the cycle, not the mention the increased chance of contamination or some other error.
Threre are a number of different methods of synchronizing cells. If you were to do a PI stain of a happy, near-confluent culture, you would see the majority of cells in G0/G1, a smaller percentage in G2/M, and some in S phase. Synchronizing cells basically gets all of the cells into G0/G1, so that you can be sure that any subsequent cycle progression is a result of your treatment and not the result of cells completing cycle progression that began in culture. Just as a caveat, some authors argue that a culture can never be purely synchronized, except possibly if you were to FACS sort by the smallest cells in the live cell gate.
As far as your drug concentration range, most investigators do titration experiments where they examine the effects of a range of drug concentrations, starting from zero to some outrageously high concentration. There is generally some concentration at which further addition of drug has no effect.
that's great help Cassio!
Thanks very much! Let's see how my expts go these few days!