I have been doing cell culture experiments that involve treating the cells with estradiol (E2). There are many papers published that state these cells are responsive to estrogen. The problem is that I have not seen a single effect of E2, so I am wondering if I am doing something wrong technically. My colleague taught me how to culture cells with E2 with a specific protocol. Here is my protocol:
1. remove vial of E2 from -80C dissolved in DMSO, 10,000X conc.
2. thaw and vortex the tube for 30 seconds.
3. Dilute to 1000X in fresh DMSO, vortex 30 seconds again.
4. Add 3ul to 3ml phenol-red free medium with charcoal stripped FBS, for a final conc. of 1X, and 3ul DMSO to another tube of 3ml medium as vehicle control.
5. Vortex medium 30 seconds.
6. Add 90ul E2 medium or vehicle medium to each well containing 5ul cells, 96 well plate format.
Should I be adding the E2 directly to each well containing cells in hormone-stripped medium instead?
Your protocol looks fine. Personally, I add the E2 straight to the plate I want to stimulate, but it shouldn`t make a difference. How old is your E2? E2 is not very stable so you might want to generate a new stock. I'm also assuming that your cells have been in charcoal-stripped FBS for at least 2 days...
Hi, my cells have not been in charcoal stripped FBS for at least 2 days because they are primary B cells and start to die after 24hrs in culture without stimuli. So, the culturing must be short term. Basically, from the moment I get the fresh blood in the morning, the cells are already in culture 4hrs later. I do use wash buffers with charcoal stripped FBS however. How many ul E2 do you add? Is it in EtOH or DMSO?
I have tested the E2 with MCF7 cells and they responded very well to E2, so I am assuming the E2 is fine. How long is E2 stable @ -20 or -80C?
should I be adding the E2 in EtOH directly into each well of cells or into the culture medium followed by adding the medium to the cells?
In my esperiments I prepare the desired volume of medium with solvent and treatment sufficient for all the wells and then I change medium replacing with treatment medium
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