NHS Greater Glasgow and Clyde
Question
Asked 10th Sep, 2014
How much FBS do I need to add to the medium in order to prevent cell division in 96-well plates?
My MCF-7 cells are dividing in 5% medium, but I need to keep them alive and prevent them from division...
Most recent answer
I would carry out a serum titration i.e. 0.1, 1, 2 and 5% and measure their proliferation using either cell count or SRB assay perhaps or even Cell Titre Glo. This would give you your best answer and also allow you to assess whether the cells can tolerate low serum (although as above MCF-7 can).
Regards
All Answers (4)
University of Melbourne
May be you could try reducing it to 2%. I have grown primary cells with 2% FBS earlier and they stay alive, I suppose it should work with cell lines too..
1 Recommendation
Medical College of Wisconsin
MCf-7 cells are estrogen receptor (ER) positive and typically tend to thrive in medium with even low levels of estrogen or other estrogenic compounds. FOr example, phenol red which is added to most red-media to indicate the change in pH is estrogenic and can affect the growth of many breast cancer cells (see B.Katzenellebogen's work from the early 90's). Similarly regular FBS also has many growth factors in addition to estrogen. For these reasons, in order to truly estrogen starve MCF7 cells (and this might be the best way to reduce cell division without adding other compounds) one needs to use charcoal stripped serum, which uses carbon binding to estrogen to reduce the estrogen content of the serum. Also it will be wise to use pheno-red free (white) media. That said, MCF7 cells are notorious for binding tightly to estrogen, even when present at low levels, so that it may take a couple of media changes to reduce the estrogen content.
I have successfully followed the following protocol to cause cytostasis in MCF7 cells.
Grow MCF7 in regular media (in my case DMEM+5% regular FBS). On Day 0 remove red media, and wash 1X with Phenol-red free DMEM+ 5% Charcoal Stripped FBS. Replace media and allow to sit in incubator for 2-3h. Repeat this proceduce every 2h at least 4-5 times on Day 0. Leave cells overnight in white medium. You can continue this for one more day if you want to make sure all estrogen is gone. Alernatively, you can plate these cells in 96-well plate, again in white media and proceed with other experiments.
1 Recommendation
French National Centre for Scientific Research
I also think that you could reducing it to 2%. I have maintained HepG2 and SkHep1 cells in basal medium supplemented with 2% FSB, when I need a low rate of cell prolifetion.
With 2% FBS you can increase the "time needed" to cell division at cell culture, like a slow growing
regards
NHS Greater Glasgow and Clyde
I would carry out a serum titration i.e. 0.1, 1, 2 and 5% and measure their proliferation using either cell count or SRB assay perhaps or even Cell Titre Glo. This would give you your best answer and also allow you to assess whether the cells can tolerate low serum (although as above MCF-7 can).
Regards
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