working with hormones - what is the stock concentration, etc. (Jan/19/2006 )

Hello all,

I am new to cell culture and no one in my dept has worked with hormones before so I am completly out of my element here. My question is what stock concentration people use when working with estrogen and progesterone? I need a final estrogen concentration of 10-8 and a final progesterone cencentration of 10-7. I have already tried making stocks and working solutions myslef, but the cells never expressed markers of differentiation which that are supposed to do under hormone treatment. So I did something wrong.

The flyer that came with the hormones suggested making a stock with ethanol.

Many thanks,
Vinny

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Hi,
For 17-beta-estradiol, we use a stock solution of 10uM.
Prepare by making stock solutions of 17-β-Estradiol in Ethanol and then diluting the stocks in media to the desired final concentration. Keep the stock cold (-20'C).
Different batches of cells will respond to hormones differently. In order to see a good result, perhaps you could consider starving your cells of hormones for a few days ( i do 3 days).
What this means, is you change the media from the one you're using (ie DMEM or RPMI), to phenol red free (DMEM or RPMI). The FBS you use should be charcoal stripped. After a few days in this, then treat it with the hormones, and you should see a difference.
Or you could do a concentration study, maybe your cells just need more hormones.
Hope this helps.
Vetticus

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[/quote]

Different batches of cells will respond to hormones differently. In order to see a good result, perhaps you could consider starving your cells of hormones for a few days ( i do 3 days).
What this means, is you change the media from the one you're using (ie DMEM or RPMI), to phenol red free (DMEM or RPMI). The FBS you use should be charcoal stripped. After a few days in this, then treat it with the hormones, and you should see a difference.
Or you could do a concentration study, maybe your cells just need more hormones.

[/quote]

you can pass the cells from 10% FBS to 10% FBS-(dextran coated) charcoal (DCC) stripped for 2 days, then to 3% FBS/DCC for 5 days; finally stim your cells in 1% FBS/DCC in medium w/o red phenol

Sébastien_

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Thanks for the replies.

I have one question though, why is it important to not have phenol red in the media when growing the cells before (and during?) hormone treatment?

Many thanks
Vinny

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i cite: "phenol red, which bears a structural resemblance to some nonsteroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (15-45 µ M) at which it is found in tissue culture media"

Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS.
Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.
Proc Natl Acad Sci U S A. 1986 Apr;83(8):2496-500.
PMID: 3458212 [PubMed - indexed for MEDLINE]

Sébastien_

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We make a 100 mM stock of estradiol in 100% ethanol and then subsequently dilute that to 10 mM in 50% ethanol. The 10 mM is then diluted to 1 mM in water and this is used to mkae further serial dilutions. This way the final concentration of ETOH is less than 0.01% and you do not need an ethanol control.

Agree compltely with seb and vetticus...you need to use charcoal stripped serum and phenol red free media for ascertaining the effects of the hormones. Effective Estradiol concentrations vary from 0.1 nM to 10 nM, Easiest thing to do is a concentration effect on cell proliferation.

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