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Dying endometrial stromal cells - (Sep/03/2010 )

Hello to everyone!

We do have a problem in our lab with endometrial stem cells. We are trying do decidualize them using hormones - Estrogen, Progesterone and cAMP. The problem is that cells are dying on 3 to 6 day. First we though it could be from the alcohol in which Estrogen and Progesterone are dissolved, but the control wells with just the same amount of alcohol seemed just fine. Then we though it could be from the NH4OH in which cAMP is dissolved or the concentration of cAMP is too high, since it's known it can cause apoptosis. We tested that theory as well, but cells are still dying. We tried different culture mediums - DMEM + 10% FBS or RPMI with 10% FBS inactivated. The result is that cells are dying. The interesting thing is that we have wells with nothing but medium and again cells are dying. We tried on 24 well plates or on 6 well plates, there is no difference. Another thing is cells are dying in seemingly absolutely random wells, there is no logic, and they start dying from one place of the well and it's spreading among.. The medium is clean, no contamination observed. We are culturing them in flasks as well and even when they are very dense, they are just fine. In wells we plate them in subconfluency and when they make a monolayer we start treating them with hormones. We change their medium on 72 hours since we measure prolactin, that is the only thing which we do differently from all the articles, where the medium is changed every two days. I started a test experiment today, and I am going to change the medium every other day. So far my only thoughts are they become to dense or the medium should be changed more often. Everything else we came up with we already tested.

I am attaching some pictures of dying cell from 24 well plate on day 4 and of normal cells from another well in the same plate:
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Are you sure there is no contamination? Are you using any antibiotics in your culture medium?

You could also try changing the medium every 48 hours as they did in your articles. Do you stimulate them with any cytokines to keep them alive at all?