I am trying to perform gene expression experiment with 17 beta estradiol using HepG2 Cell lines. Somehow i am not able to get any induction or akteration. It looks like i am doing something wrong in the procedure or making a mistake in making drug dilutions. I tried dissolving estradiol in 2 ways:
1. Dissolving E2 in 100 % ethanol followed by subsequent dilutions in 1X PBS, in this case i saw crystal formation in the solution may be because of PBS being insoluble in ethanol.
2. Then i tried dissolving E2 in 100 % ethanol followed by subsequent dilutions in water, no crystals were seen, but still no response was seen.
You can dissolve in combination of water and ethanol to 75 mM-80 mM
I would prepare a stock of estrogen (E2) in 100% ethanol. Then prepare sub-stocks (working concentration stock) of 1 micromolar, 10 micromolar, and 100 micromolar. Store all of them at -20 degree C. Dilute from the working stock concentrations to get 1, 10 or 100 nM final concentration to treat cells. Culture medium needs to be phenol-free and you need to use charcoal-stripped serum (to reduce basal endogenous levels of steroids, including estrogen) to get a response. Use known E2-inducible gene as an internal control. Induction of genes is E2 concentration-dependent.
Thanks sir, but for how many days i have to culture cells in phenol red free media before giving E2 treatment?
The estradiol you are using is from sigma? Check also the datasheet what they suggest you for preparation. Hope u have solved the problem by now. I am also trying to find the best conditions for estradiol incubation in cells.
See this link
http://www.protocol-online.org/biology-forums/posts/12902.html
Regards
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