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Can anyone tell me why my cells are dying? - My mouse embryonic stem cells are dying and I'm baffled as to why. (Mar/26/2008 )

I'm hoping someone might be able to shed some light as to why my mouse embryonic stem cells have been dying. This is the pattern of events;

Day 0: I thaw out the mouse embryonic stem cells from frozen stock
Days 1-3: cells appear to be fine, although growing slowly. Medium is red and clear.
Day 4: medium turns orange overnight, but remains clear. Colonies begin to detach from the plate surface.
Day 5: medium orange 7 clear. ~50% of colonies now detached and floating in medium. Colonies are visible to the naked eye as white specks, both floating and adherent.
Day 6: medium orange & clear. Majority of colonies now detached. Plates discarded.

I'm growing the cells in a basic medium of 90% DMEM (without Hepes), 10% FCS, 1:100 L-Glutamine, 1:100 beta-mercaptoethanol and 1:150 LIF. I also used different combinations of PENS (1%) and Amphotericin B (0.05mcg/mL and 0.25mcg/mL), due to having experienced bacterial and yeast infections in the past. Below are the different combinations I was using;
No PENS and no amph B
PENS 1% and no amph B
No PENS and 0.05mcg/mL amph B
PENS 1% and 0.05mcg/mL amph B
No PENS and 0.25mcg/mL amph B
PENS 1% and 0.25mcg/mL amph B

The plate with PENS 1% and no amph B was affect by yeast within a few days and discarded. The rest of the plates showed no signs of yeast or bacterial infection, but all went through the same process of medium colour change, detaching colonies and white coloured colonies. It didn't make a difference whether or not there was PENS or amph B present.

I've also made up test plates of medium and left them in the hoods while I've been working. The first one I did became infected with yeast. I switched hoods and made up two more test plates, one for the original hood and one for the new hood. These haven't shown any results yet (day 2).

Has anyone experienced something similar with their cell cultures?
Please help.


this may be a silly question, but when you found that the hood you were working in had contaminated your media, did you throw away your media and make up a fresh batch?


I didn't at the time; I was pretty sure it wasn't the medium, as other plates with the same medium in them hadn't become infected with yeast, despite not having any anti-fungal angent (amphotericin B) in them. Since then I've finished the medium and have made up more, with no difference in the condition of my cells. Furthermore, I was using two different batches of medium at the time (one without PENS and one with PENS) and it hasn't made a difference to the cells which medium them were in. So I concluded that it wasn't the medium that was the problem.