Long-term drug treatment of fibroblasts - (May/19/2010 )

Dear guys,
I have to treat fibroblasts w/. a few different drugs and analyze the effects of each treatment by EM and western blotting.
My plan is to grow my cells on 6-well plates and analyze the effects at several time-points over ~1 month.

I would appreciate your advice on my experimental design. I was planning to have time-points T-0, T-5hrs, T-3 days, T-1week, T-1.5weeks, etc.
I was thinking of keeping 2 wells per time-point (for each treatment), such that one well would be devoted for the EM analysis and other one for the Western Blotting.
Further, I was thinking of starting T-0 at confluence (T-5 hrs would use adjacent set of wells and would also be at confluence), and then changing the media and splitting the cells w/ trypsin as needed for the other time-points. Does this seem reasonable?
Do you think that maintaining the cells by trypsinizing them, for the later time-points, would interfere too much w/ the specific effects of drug treatment I am looking for?
I thought this is something I had to do since otherwise the cells in each well would overgrow. Basically, the trypsinization and splitting would be the only manipulation of the cells other than changing the media.

I am also thinking about how often I would have to change the media for each drug treatment, since the drugs have different half-lives. I believe for one of them I would have to change the media daily.

Does this seem reasonable? Thanks in advance for your suggestions!

cell-culturer

I would say that your biggest problem is going to be that at the end of the experiment you are going to have a huge number of plates of cells to harvest if you keep them all.

Many drugs do not work effectively on cells that are confluent as many of them rely on active division for their action, though this does vary according to the drug. Also some fibroblast cell lines are contact inhibited, so once they are confluent they stop growing, which may alter how the drugs behave.

Because you will be passaging cells, you may be best to harvest your 0 time point at an average density (say 70%) rather than confluence, so that it best represents the state of the treated cells during the experiment.

thanks so much for your help!

bob1 on May 19 2010, 06:57 PM said: