I used to work with MCF-7 cells and a murine mammary cell line LM05-E and studied effects of estrogen and tamoxifen. We always did it the way you said, we grew cells in normal medium until experiment, we starved cells (medium without serum) for 24 hs and then treated with medium with charcoal stripped serum 1% supplemented with estradiol and/or tamoxifen for 24 to 72 hours and measure proliferation, cell death, western blot, mammosphere forming capacity, etc.

I believe that if you grow your cells all the time with charcoal stripped 1% the will grow to slow. Growing them with normal condition and starving before experiments works well.

Hope it helps

Thanks for your reply Diego. What do you think about the time of starvation? Is it enough 24h of starvation to retrieve all the conditions of the normal medium? Certainly, the growth on medium with stripped serum makes the cells growth slowly (to much slowly some times....) But... must this procedure be better to avoid all previous effects of hormones present in the normal serum? Let me know your opinion.

It worked well for us, longer starvation could affect cell viability depending on the cell line. If your cells can handle 36 or 48 hours starvation you could use longer starvation. However it depens on what you want to study, if you want to analyse viability or proliferation it may not be advisable to starv for longer periods. Some cell lines start dying at 24 hours. We tested 24 and 48 hs starv and cells responded as expected to hormone addition.

Thank you again Diego. For the moment, the starvation strategy seems the most adequate to work with E2, P2 or other related compounds. We will see if other colleagues agree or propose additional and/or topic specific strategies.

Dear Diego, some questions arise... why to use 1% charcoal stripped serum after starvation and not 10%, if normaly cells growth in 10% of FBS? Another question, have you detected that cells under starvation do not attach to the plate?

In our experiments what we were interested to know was the response to estradiol or tamoxifen. I believe that the idea of 1% serum is that cells will not grow with that concentration alone, so the growing effect that you see or death will be due to the drugs you are treating with. Is a condition in which cells survive but need other signal to grow, at least that was the case in our system, you will have to check if 1% is enough for your cells. If you use 5 or 10% cells will grow due to the serum and you will not be able to see the effects of your drugs.

Cells will not attach without serum, you need to grow them first with serum until the required confluence that shoudn´t be so low, try 40 or 50% if not cells will die during starvation. Once attached and growing they will survive starvation. However, keep in mind some cells are very sensible to starvation.

Hope it helps!

Thank you very much Diego for your answers. One question more. How important is the step you perform in your experiments regarding to washing with PBS five times?

In those experiments that were short pulses of 15 min or 30 min it was necessary because we wanted to really clean any traces of hormones, for larger experiments of 24 or 48 hs culture with hormones we used only 3 washes.