We are going to perform growth curve , cytotoxicity assay,protein estimation,ELISA, Anti cancer drug (Tamoxifen) effect on cell population and Soft agar colony Assay. We are using without Phenol-red media for that. Because Pheno-lred is having Estrogenic effect and we are going to want to see anti-estrogenic effect of Tamoxifen on MCF-7 cell line. But the cell growth is not proper in without Phenol-red media EMEM(Sigma). Cells are always compile on each other and not properly adhere on the surface of Culture flask on 2nd or 3rd Day (after passage). We can not get more than 60% confluency .
In short,We are in trouble in establishment of MCF-7 cells in without Phenol-red media EMEM.(Sigma)
So what should we do?.
Dear Dr Khushali Upadhyay
No problems, I cultured the MCF-7 cells many times without phenol red but I was use RPMI-1640 medium + 10 FBS
I think you must change type of media
GOOD LUCK
Phenol red acts as an indicator of pH change (from neutral to acidic), so even if you use media without phenol red, it should be completely fine for the growth of your cell lines. And I agree with Serag Eldin (RPMI-1640 with 10% FBS)
Hi. FBS is full of estrogen (and other hormones) so you should more worried about this estrogen rather than the phenol red activity. if you really want to see the effect of estrogen and tamoxifen in a very clean assay, you should use Phenol red free DMEMF12 + 10% charcoal treated FBS. In all the case, you should put 10-6 to 10-7M of 4OH-tamoxifen in your test and you should be in a very higher range than the physiological level of estrogen from your growth media, so just by competition you should see the tamoxifen activity rather then the estrogen one. I will advice you to just use the normal media and add tamoxifen to mimic the human condition to ensure happiness of your cells.
In our assay,we directly treat the cells with Tamoxifen,which is dissolved in Absolute Ethanol having Concentration range: 10^-6 to 10^-10 which is Same in 4 OH Tamoxifen. Is it ok or not??
tamoxifen or 4hydroxy-tamoxifen? The tamoxifen is the non active form that need to be treated by the liver (to be hydroxylated) and then it is active for in vitro assay... we use 10-6 or 10-7M.
we are using Tamoxifen and 4 OH Tamoxifen ,both is Individual treated to MCF-7.
The Concentration of both solutions decided by MTT Assay. That is 10^-6 for Tamoxifen and 10^-8 for 4OH Tamoxifen. In these treated cells,we will going to check the elevated level of Cellular TGF beta-1 and compare it with non treated cells.
Hello
I grow MCF-7 in RPMI 1640 . add , i cultured MCF-7 cells in
DMEM to 50mL
FCS (10%) 5mL
Sodium pyruvate (1mM) 500uL
Insulin (10 ug/mL) 500uL
Non essential aa (0.1mM) 500uL
Antibiotic/mycotic (1X) 500uL
With 10nM estrogen, there was about 3-4 times more cells than without estrogen. , but there was a huge fold increase.
- Some media recipies recommend adding estrogen. If the media you're using is phenol red free, this also reduces the cell growth rate, because the chemical structure of phenol red is similar to estrogen, and the cells respond accordingly.
Good Luck
We normally use Phenol red free media (EMEM) and charcoal stripped FSB for our MCF-7 cells.
Alkarim sir,thank u.. But we want to check the effect of Antiestrogenic drug,Tamoxifen on cellular TGF beta level in Breast cancer. So we should use Phenol free media ,estrogen or any growth factor free media.
Dear Dipak K Banerjee
I also tried to grow MCF7 in pnenol red free(DMEM) and charcoal stripped 5% FBS, but after 3-4 days of growth all the cells detached. What other factors should i concerned for ?
Who is the supplier of FBS? Second, to maintain a stock culture you may use regular EMEM with phenol read and unstripped serum. But, depending upon your experiment you may switch to phenol red free EMEM plus stripped serum for a short period of time. I hope it helps
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