I transfected J82 bladder cancer cells with pmep4 vector, and thereafter performed selection with 300 ug/ml G418 (the optimal concentration had been titrated beforehand). During the first week, I saw several isolated colonies that were growing well, but starting a bit after two weeks, most colonies, that had earlier been growing during selection, started dying. As of now, I only have two colonies left, and these are neither growing nor dying. The only remarkable thing about these cells is that they seem pretty big. As concerns bacteria, the cells are mycoplasma checked, and there are no signs of any other contamination. Three days ago, I changed medium to G418-free to see whether that would make the cells grow, but nothing has happened. Any suggestions as to what could have gone wrong, what I can do to save the cells and any additional things I should do during a new transfection?