Hi,
I am currently culturing 4 different cell lines Vero, BHK, JEG3 and BeWo.
This morning I noticed that in all my flask, I had huge numbers of dead cells that had clumped up and sort of floating around in the medium.
Medium looks pink and not yellow like one would expect if there was a contamination.
I checked the CO2 by fyrite - and it is at 5%, temp reads fin by a thermometer in the chamber.
There is water in the pan at the bottom so humidity should be fine?
I have a Fisher Isotemp incubator...
Right now I will move all my cells to different incubator to make sure there is nothing wrong in my media...however the FBS has been unchanged since 3 months so that should not be a problem
I did however open a new bottle of DMEM for the Vero, BHK and JEG3. This however does not explain why the BeWo died too - since they use a diff medium F12K....
I am desperate - please let me know what you think could be wrong and any suggestions that you might have.
Thanks in anticipation!

Try staining with DAPi, and look for small spots on the periphery of the cells- thats a hallmark of mycoplasma infection (which will not turn your medium yellow).
However, that said, its very unlikely that it will simultaneously infect very different cell types in different media... Do you use disposable pipettes, or reuse pipettes? Could it have been some otherhandling isse?
Also, was the temperature steady at ~37 - temp of 42dC can cause massive cell death if it remains for more than a few mins.
Finally, ensure that nothing added to the cell contained stuff like DMSO, proteasome inhibitors etc - weird things have been known to happen!

Thanks for your response.
DAPI staining looks fine.
I double checked my FBS stock -turns out a month ago, we switched to Mexico qualified FBS from invitrogen ..previously we used the USA certified one.
I wonder if that could be the cause??
But why would it happen after a month of the switch?
I don't add anthying to my media except FBS - no Pen strep either...

I will also check if the temp is steady at 37 - it seems it is with the visual display but I need to check with the thermometer over time.
I am not sure if I should already thaw new cells from my stock....

Do you know if the CO2 tank has been changed recently? And does it supply only the 1 incubator? If the cells do fine in a different incubator on a different tank of CO2, there might be a problem in the one tank. Typically you want medical-grade CO2; industrial grade, while cheaper, can have some odd contaminants. I have to agree that the change in FBS is not likely to be the source of the problem; I would have expected that the cells would have adapted to the new serum after a month. If you have access to a recording thermocouple or a chart recorder you can check to make sure the incubator is maintaining the correct temperature over the course of a day. Manually reading a thermometer makes it a lot harder to catch transient temperature spikes.

The Co2 tank has not been changed in the last 6 months.
It still has enough..I am not sure what the rate of consumption should be and how often it needs to be changed.
I will double check on the quality of the CO2.
Anyhow this morning, the remaining of my cells also sloughed off completely so I trashed all my flasks.
I did have them also in another incubator supplied by a different CO2 tank and those too had the same outcome -i.e. dead :(
I will try and compare the USA certified and the Mexico FBS just with one cell line that I will freshly thaw and then hope the answer lies therein.....
I hate this as I cannot continue with the intended work with these cells.....but what can I say - that is what science and research is all about!
Thanks for all your help and ideas.
If you have any other thoughts, please do post them.

I'm sorry to hear that.
One suggestion: when you thaw your next vial, can you do it in parallel in your lab, and somewhere else, using the other lab's reagents/media/incubator?
It will help you localize the problem - i.e. if its just your lab. Are other lab members' cultures also experiencing these issues?

Unfortunately, I am the only one who uses cell culture in our lab.
Yes, that is a very good suggestion - I will thaw 2 vials and use them with other lab's media and at diff locations....

Thanks a bunch!

Hi,
I just noticed that my CO2 tank expired in August! Does this have any bearing on the environment of the incubator?
I am guessing it must - I will check it out with the company that refills for us. Meanwhile thought to share with this group in case you may have had the same encounter...

I think that you have to see if in your culture you have Hepes buffer or sodiumk bicarbonate. In our lab, we always make the folow:
1. we make the media
2.We measure the pH. if is to high we decrease to 7.4.
3. Then we add sodium biocarbonate to avoid the changes in pH.

I think your incubator is Okay. We have the same problem. If you want you can contact me later this weeek and I can give you the concentration of sodium bicarbonate