A few questions:

Did you generate this cell line yourself? If so, how did you freeze back the cells? Your choice of freezing medium or protocol could have reduced the viability of your cells.

If you generated this cell line, did you generate this cell line via clonal expansion of individual cells in tamoxifen conditions, or did you grow a population of MCF-7 cells in tamoxifen? If you selected against a MCF-7 population, it could be the case that the tamoxifen-resistant cells did not survive the thawing process as well as the tamoxifen-sensitive cells.

If you obtained this from another lab, do you know what conditions they were grown in? If you're growing the cells in a different type of medium and/or FBS, they could be experiencing shock from different culturing conditions.

On that note, if you're trying to switch tamoxifen-resistant MCF-7 cells from phenol red and regular FBS to estrogen-deprived conditions all at once, that could be negatively affecting their viability. Whenever I adjust breast cancer cells to estrogen-deprived conditions or any cell line to new medium and/or FBS, I transition them gradually.

This is done by incubating them in a series of 75%:25%, 50%:50%, and 25%:75% ratios of old medium to new medium for 2-3 days each. After that adjustment period, I keep them in the new medium for at least a week before experimentation, and save any excess cells that result from passaging by freezing them back.

hey thanks,

yes i generated them myself in a year ,growing in a population of MCF-7 cells in tamoxifen.

yes i understand the shock treatment and i slowly transferred them,thou i remember that in CSFBS they were only two week solder.

so i switched over to freezes of Phenol red free medium,which were generated by a long period of survival from phenol o red medium.

But the freezes are not surviving when ever i put tamoxifen the cell debris and floating cell s occur.

now if i passage say one passage without tamoxifen they survive.

so that means my freezes are not bad.

any further guide how to revive,can i thaw them in phenol red medium with insulin,see if they survive then,slowly change to phenol red free medium?

thanks

In my experience its hard to freeze the cells and used them again as a matter of fact the cells have survived as a clonal selection due to the permanent drug exposure ,so if you cease them or freeze them .simply they lose the properties they already acquired .its kind of annoying to deal with these types of cells because you most always keep them in the same condition ..good luck

Thanks

I am a little confused about the exact conditions that you used to generate the tamoxifen resistant MCF-7 cells. There are a variety of conditions that change the nature of their resistance. Were the cells grown with estrogen and tamoxifen, or without estrogen, but in medium containing tamoxifen? As tamoxifen binds to the same ER as estrogens, it must be noted that the binding is at 1000-fold lower affinity. Estradiol can reverse tamoxifen inhibition at a much lower concentration.

If the cells were grown in estrogen free medium but had phenol red in the medium, the effects of estrogen are further compounded. I would expect that you might have used phenol red-free medium supplemented with charcoal stripped fetal bovine serum.

Many subclones of MCF-7 have been established. They have peculiar variations but yours would seem to be more similar to a line designated MCF-7:5C. These were developed by long term estrogen withdrawal. However, addition of estradiol triggers an apoptotic response rather than enhancing growth. This is not quite what you describe, but reminds me of your reference to the occurrence of floating cells.

My understanding is that the MCF-7 LCC subclones (-LCC1, -LCC2, -LCC9, etc.) were generated to illustrate the various attributes of tamoxifen-resistant cells. I do not know as much as I would like about these cells, but it would seem the LCC1 represents an estrogen-independent, stable cell line that is still tamoxifen sensitive.

I guess this does not provide much of an answer to your original question, but I always feel like certain questions do not provide enough information for me to begin to formulate an answer. May I recommend the following manuscript for much useful information: Horm Mol Biol Clin Investig. 2012 February ; 9(2): 143–163. doi:10.1515/hmbci-2011-0004. Though it does not answer your question, it provides a wealth of information about acquired antihormone resistance.

Thank you for providing a very thought provoking question. It has me doing a lot of digging.

ya ,i am generating them initially with phenol red DMEM then, with phenol red free DMEM an dtamoxifenand initially used MCF-7 from USA origin.then i switched over to Charcoal stripped fbs.

we haven't added up any estrogen.

now everything was fine,my freezes ? i am not able to revive them as soon as i put tamoxifen in phenol red free DMEM ,the cell debris i sthere,i trypsinized them, and re-plated but i can separate cell debris from cells.

i have less freezes left now,any way to overcome cell debris?

thanks for answer

you mean to say tamoxifen is still sensitive to cells,but before they were growing in culture happily and showed 90% survival by MTT assay as well. maybe there is till some sensitivity but why cell debris now? how to over com ethat?

We add insulin as well 10ng/ml

so growth should be proper

I think freezes are not good as well?

Http://www.ncbi.nlm.nih.gov/pubmed/10070856

The question is related to my previous discussion of resistant cell line mcf-7 tamoxifen,even if i take good freezes which were growing nicely before,tamoxifen is still killing cells.cell debris lots that cells are growing but debris takes the space.i checked media.it is fine in incubator.tryspisn and pbs works finefor other cell lines,then?how come freezes are not good all of a sudden.need guidance.thanks