Please, how do i create a tamoxifen resistant MCF-7 cell line? how do i calculate the drug concentration and number of cells?
At the first glance, this seems to be an easy question, however, up to now, there are many protocols around that vary in concentration, tamoxifen derivative as well as incubation time and media conditions. So, I think there is no generally accepted "gold standard" found yet and the resulting tam-resistant cells may also vary according to the protocol used. Nevertheless, there are some features that are shared by virtually all protocols: It will take about three months until the MCF-7 cells do not care any more about the presence of the drug.
4OH-tamoxifen is mostly used because it is more potent than tamoxifen itself, which is slowly converted to the 4OH by the cells anyway. 1 µM 4OH-Tam is a dose often applied, but ER-blockage will be efficient already at 10 nM. The cells will also continue to divide slowly with tamoxifen present, because other factors, especially from the serum become alternative proliferative signals. So it is not just adding the tamoxifen, removing dead cells and wait until some resistant cells appear with a certain frequency. You will have to transfer the cells to new flasks on a regular basis. Most groups also use medium without phenol red as is has some estrogen like properties.
I hope that helps a bit!
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