I am checking the mRNA expression of estrogen receptor along with other genes following estrogen treatment in MCF-7 cells. But I am treating cells with 100nM E2. Is it a very high concentration for 1 x 106 cell density? Also I am culturing MCF-7 cells in phenol containing media first and after 24h, changing the media to phenol red free media + 5% charcoal stripped FBS for 6h followed by treatment with E2. Am I doing appropriately? So far I am not able to see a significant change in the mRNA expression of ESR and other genes as well as no change in proliferation rate when compared to untreated cells. Please suggest. Thanks.
In my experience, MCF-7 cells (1*106) treated with 10-12,and 10-9 M E2 showed significant changes at mRNA after 24 hours. but my culture conditions are RPMI phenol free media+10% charcoal stripped FBS.
You should culture your cells in hormone free medium rather than change medium after 24 hours
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I think one should standardize the dosage of estrogen treatment and obtain a significant dose response curve and accordingly design an experiment. The concentration will differ on the cell types used and also on the type of response you need to measure. So, for your own experiment, you need to investigate in this manner.
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