DG44 CHO cells and Methotrexate - (Sep/19/2011 )

Hi, I am new here but I haven't been able to find an answer to this question anywhere else. I am trying to do gene amplification in DG44-CHO cells (using Invitrogen's pOptiVEC-TOPO vector.) The protocol from Invitrogen says I should start with 50nM Methotrexate (MTX) and there should be some sort of die off of non-adapting cells--followed by a gradual return to ~90% viability as those cells that adapt expand in the culture. Once the culture returns to ~90% viability the round of MTX is considered done and I can freeze an aliquot of cells and move up to 100nM MTX and so on up to like 4uM MTX. That is how I think it is supposed to work--but when I tried adding 50nM MTX to my cells nothing happened. After 3 passages at 50nM I upped it to 100nM and still saw nothing. I then set up one set of flasks to keep passaging at like 300nM and another set that went up to like 10uM. At both 300nM and 10uM I didn't see a huge drop off, but both are growing much slower and the 10uM has over the course of a couple passages droped to like 50% viability. My question is this: DId I just get impatient and should have kept them at 50nM for the whole 3-4 weeks? Or did I do the right thing by keep upping the MTX until I saw an effect? Has anyone else done MTX amplification that can tell me what the cells did right away and what they looked like throughout? This has been driving me nuts and Invitrogen hasn't been too much help so far. Thanks for any help you can offer!!!!!!!!!!!!!!!!!!!!!

Never used methotrexate, however:

Try using charcoal stripped FBS to get rid of the folate in the medium. I would use the whole incubation period, these sorts of things aren't ones you can push in terms of time.