Hi all,
I am using 10nM of Estradiol (E2) on MCF-7 cells but can't observe a stimulating effect from E2
cells were first cultured in normal medium then seeded (104/well) in 96 well plate in medium (containing phenol red) without serum, and treated with 10nm E2 or vehicle(ethanol).
however I can't find any difference between E2 group and vehicle group, is there anything wrong?
I am expecting higher cell count(visually)/viability(WST1) in E2 treated group.
E2 were dissolved in absolute ethanol at 0.5mg/ml then diluted to 1000x stock solution with medium,
shall I prepare E2 in 1000x stock solution with ethanol instead of medium?
Meanwhile, I am using 1uM hydroxytamoxifen (4-OH-TAM or 4OHT, dissolved with heating, 1000x stock solution in ethanol) on the same MCF-7 cells supplemented with 10nM E2, no difference was observed neither.
however, the 1uM 4OHT seems to have stimulating effect on MCF-7 cells cultured in the same medium with 10% FBS and 10nM E2, which is very confusing.
anyone has any idea what went wrong? Thanks in advance!
P.S. the same MCF-7 cells were treated with tamoxifen (TAM) at 50uM/100uM in 10% FBS supplemented medium and have observed excellent suppression in cell growth and apoptosis. so I am quite sure it's not a problem with the cells. I just can't get the 4-OPT to have the same effect as TAM.
Update1: I have used Charcoal-stripped serum and the Estrogen have worked as expected, now onto 4-OHT...
Update2: well the 4OHT is actually working as expected (in CSFBS and phenol red-free medium, with 1nM E2), measure the cell viability at 5-7 days intervals after the treatment and the inhibiting effect 4OHT will show.
Hi Xiao, We have also had confusing results when tried to establish MCF-7 proliferative assay. I would suggest you to spend some time for optimisation of this method, namely
1) solvent used for E2 (and your samples), I have good experience with methanol. But DMSO and ethanol should be also fine, according to literature.
2) test more concentrations of E2 (serial dilutions around EC50s found in literature), lets say 10pM, 100pM, 1nM, 10nM...
3) amount of cells plated per well - this number differs a lot in literature.
4) cells passage number and general fitness - when we tried to establish this assays, cells were growing very slowly, we had to find optimal procedure for maintaining, and passage nr. for testing - generally 10+
PS: i think your 1000x stock in ethanol should be fine, as I use 200x stock in methanol.
Have luck!
J
Xiao,
You didn't indicate if the FBS had been stripped of hormones or not. If not, then it is probably loaded with estrogen and progesterone at the very least. This can obscure the effects of added E2 or other hormones. You can deplete FBS of steroid hormones using activated charcoal or buy it depleted. Any experiments done in vitro to study the effects of steroids should be done using stripped serum.
Good luck!
Hi Jakub, thanks for the input, which are very helpful.
As for the E2 solution, the sigma-aldrich website says 1mg E2 in 1ml ethanol, then add 49 ml medium, I am not using that much medium, and weighing small-mass quantity as 1mg is not that accurate on the device I use, so I dissolved the E2 at 0.5mg/ml in ethanol then diluted to 1000x stock solution (10uM) with RPMI 1640 (without serum), I am suspecting that I should have diluted the 0.5mg/ml solution in ethanol instead of medium for the 1000x E2 stock solution, then add to the culture. The same concentration of ethanol in culture seems to have minimum effect on cells. Will repeat the experiment with ethanol/methanol solutions (and as you said, in broader spectrum of concentrations) .
I am still confused about the 1uM 4-OHT's stimulating effect on MCF-7 cells, yes we have observed the stimulating effect of low dose TAM, but from I read on literature, 4-OHT should be a absolute estrogen receptor antagonist, while TAM is known as estrogen receptor agonist (low) and antagonist (high). will repeat this too.
Regards!
Hi Robert,
Thanks for replying, the charcoal stripped FBS we ordered are still on the way, that's why I used serum-free medium for testing E2 property, which is not working at the moment, do you know if E2 have to work with serum (charcoal-stripped or not) to function properly? if so, E2 may not have the expected effect in the serum-free cultures.
Cheers,
Xiao.
Xiao,
I noticed that you used serum free media for the E2 assays. That was a good idea to avoid the hormones present in serum. However, you will see little or no cell division in the absence of serum, so cell proliferation studies will be inconclusive. Most cultured cell types will survive in the absence of serum or in low (0.25%) serum, but they won't divide.
RW
Robert, Thanks for the prompt response, guess I have to wait for the charcoal stripped FBS to see if my E2 solution works.
Meanwhile, do you think the phenol red in the medium can mask the effect of E2? I have read that phenol red act as weak estrogen. link here: http://www.ncbi.nlm.nih.gov/pubmed/3458212
Xiao.
http://www.ncbi.nlm.nih.gov/pubmed/3458212
Hello Xiao. In vitro experiments regarding sex hormone and/or hormone antagonist effects must be done with charcoal stripped serum and phenol red-free medium. Both the own hormones on FBS and/or phenol red really interfere in cells. Furthermore, you must also use phenol-red free trypsin to detach your cells.
used the ethanol diluted E2 in serum-free medium, no effect either, so it's not the dilution method.
4-OHT has suppressive effect on cell growth in serum containing medium but not serum-free medium, so its function is estrogen-dependent I think. serum-free medium will not work for this experiment, waiting on the charcoal stripped FBS.
In serum containing medium, lower than 1uM 4-OHT treatment had boosted MCF-7 cells performance slightly in WST1 essays if I measure A450nm at 2hours, but at 4 hours they are no different from vehicle-treated cells. 1uM and higher concentrations have significant suppresive effect on cell growth, precipitation was observed in 20uM and higher concentrations of 4-OHT. It seems 4-OHT is working as expected, but I don't think it can be regarded as absolute estrogen receptor antagonist, but it's better than TAM which as significant stimulating effect at 10-20uM.
Hey Xiao,
Any updates on the E2 and 4-OHT effects after using them with Charcoal Stripped FBS?
Thanks. Rishabh.
Hi Xiao,
I wonder if you found the answer for your question? I'm working with E2 too and I haven't been able to see an estrogen effect. I used charcoal-stripped FBS and phenol red free media from the beginning but still, no hope. I haven't tried serial dilutions yet but just want to ask if you came up with any idea.
Tamoxifen is actually a partial antagonist. So, in the presence of E2 it will inhibit, but in the absence of E2 it will activate the ER.
Hi Rishabh and Nguyen, sorry I didn't notice earlier, the E2 is working as expected in charcoal/dextran-stripped FBS and phenol red-free media. However, 4-OHT is still having stimulating effect at lower concentrations. Maybe I should estrogen-starve the cells for longer to see absolute inhibiting effect?
For Nguyen, are you using commercially available CS-FBS? if so, the serum maybe the problem. You can try charcoal stripping the serum yourself (the reagents are cheap and available from sigma), or buy from a legitimate distributor.
Yes, I think you should serum starve your cells before treating them with 4-OHT, also include phenol red in your media, for some reasons this increases the efficacy of 4-OHT.
Hi Rishabh, it turns out the 4OHT is working as intended, just give it a few more days to show, the inhibiting effect of 1uM 4OHT is very contrasting compared to control on day 7 (4uM will give you about the same effect on day 3, but I see 1uM in most literature).
currently the cells are cultured in CSFBS for 48h before 4OHT/E2 is added, estrogen-starving the cells for longer may further lower the 4OHT dose required and let the inhibiting effect to show earlier, will update later.
BTW, it will be interesting to check the effect of phenol red on 4OHT but I think we should use the phenol red-free medium since phenol-red may cause unwanted disturbance on ER, right?
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