Actually i am facing a problem that when i used drug about 1ug/ml concentration on MCF-7 and MDA, it reduces almost 70-80% population. But remaining 20-30% cells took almost 20 days to increase cell number upto 50-60% may be. during these days I have changed media for every 2 days with same drug concentration. I didn't transfer cells to the next flask because cell number was very low. but after 20 days cells got contaminated (Bacterial and Fungal) for being so long in a same flask. So what would you suggest, for that problem ???
What kind of drug do you use? maybe it causes the DNA damage to cells and senescence, therfeore they need time to overcome that state. Maybe it would be good to reduce the concentration of the drug and add antibiotic and antimicotic agent to the medium (e.g from Gibco) to prevent contamination in long term experiments. In fact how fast the cells became resistant for the drug is dependent on the drug and type of cell line, sometimes it takes a really long time to develop drug resistant cells.
I have added antibiotics pen/strep as recomended in media. Can i split the cells with trypsinization before increasing the dose of drug.
We have selected for drug resistant lines by gradually increasing the dose of the drug, starting with a dose that only slightly inhibits growth. You can do a standard growth inhibition assay such as MTT or MTS to help you select a starting point. Another approach is to do single cell cloning in the presence of the drug at the desired concentration. Single cell clones are usually more stable. The standard way to do this you simply dilute the cells to 1 cell per ml and plate 100ul/well in 96 well plates, which yields one cell per 10 wells. However since the drug will likely kill most of the cells you may wish to try 100 cells/ml and 10 to 20 plates. It shouldn't be necessary to change the media in the plates especially if you use a stabilized form of glutamine (such as Glutamax) plus HEPES buffer in your media. Once you find wells with viable growing cells you can expand them. You may want to omit the drug on the first passage from the plate, but then add the drug once the cells are growing. With single cell cloning you should always select several clones to compare. I would be cautious about relying on too many antibiotic and antimicotic drugs since they sometimes only mask the contamination, you should grow the cells without these drugs after the selection to make sure they are free of contamination. You may also want to get them tested for contamination, especially mycoplasm since it is usually hard to spot.
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