My research is to analyse the estrogenic activity of possible substances that migrate from plastics using the E-screen test based on the ability of MCF-7 cells to proliferate in the presence of oestrogens. However, so far I have not been able to validate the assay since I have failed to see significant differences between the effects caused by my positive control (17β oestradiol at almost any concentration) and my negative control. There is one paper that suggest that different "sublines" of MCF 7 cells show different sensitivities to oestrogen. So, I was wondering if you have been able to see differences between the proliferation in the presence of E2 and in presence of the negative control. and where did you get your mother stock of MCF-7
I can't answer your direct question, but it might also be worth checking your conditions carefully. For example, most cell culture media comes with phenol red, which will bind to the estrogen receptor. So you might want to just look at your various culture conditions, and make sure none would interfere with the readout.
am maintaining my cell stock in DMEM with phenol red but with every pass I do serial dilutions of the cells in DMEM w/o phenol red and supplemented with 5% Charcoal stripped FBS to starve the cells of oestrogen like compounds for at least 3 days. When the cells are 80% confluent I plate them in 96-well plates at different seeding concentration to find the concentration that works better (4000, 2000, 1000 and 500 cells/well). I add serial dilutions of oestradiol (from 10-12M to 10-6M) and compare them with my vehicle PBS.
I have also tried to incubate the cells for 4, 5 and 6 days to determine the optimal period of incubation.
I am using different assay to determine proliferation AP assay, MTS assay and DNA quantification assay.
I have also try different cell line stocks of MCF-7 cells because there are some articles that argue that different MCF-7 stocks show different sensitivities to oestrogens. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1519213/
Maybe my mistake is how I have prepared the stock of oestradiol. I did serial dilutions of oestradiol in absolute ethanol to yield a concentration 10-3M and then the serial dilutions were done in PBS.
E-screen MCF-7 oestrogen dependant proliferation assay shows no differences in proliferation between my vehicle control and oestradiol. Available from: https://www.researchgate.net/post/E-screen_MCF-7_oestrogen_dependant_proliferation_assay_shows_no_differences_in_proliferation_between_my_vehicle_control_and_oestradiol [accessed Jul 10, 2017].
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