We have a 3t3l1 cell line---never had problems before but all of the sudden having issues with phd of media going off. Using same base media on primary rodent preadipocytes with no issues...media looks normal pink-red color in millipore bottles but shifts to orange and sometimes even yellow after only 24 hours on cells. Only thing I can think of is that they are excessively metabolically active....but why? They are not changing to adipocytes despite induction likely because of pH stress. Any suggestions? Incubator is ok bc other cells in incubator and their media are fine. Remade media and triple checked that pH was correct when being made. Thoughts? Help!
Are you sure the culture is not contaminated?
A dramatic change to yellow color sounds like bacterial contamination.
Hi Annie,
As say Max one possibility is the presence of contamination in the medium. As you comment another is a higher metabolic activity in the cells or a elevated number of cells in the culture. You need to observe this parameters to know what it is happening.
Good luck,
Joan.
Sounds like they are contaminated with something. Could be bacteria or possibly a more metabolically active cell line. Are you growing any other lines?
Also is it just this batch of cells or have you thawed another vial and seen the same issue? Sometimes it is not worth the troubleshooting and will save time and stress to thaw a new vial. There are times cells simply stop acting as expected. As your other cells are acting fine it may be the best course of action for you.
I do not see active fungal or bacterial contamination and our cultures contain anti-mycotic/antibiotic. However, I read that mycoplasma will infect cells and since it is intracellular you cannot really see it...not sure if that would cause media yellowing/acidity too. This is actually 2 separate reps of cells from this line that are doing this and they were thawed from two separate vials but the same overall original stock of cells (just different passages)....however, we have used cells from that same original stock of cells multiple times with no issues similar to this. I am about to pull the plug on them but I hesitate to jump right into another rep without any idea on what is going on lest it happen again. Thanks for suggestions so far!
Hope is not the case, but if you want to check it...there are several commercially available kits for mycos. Good luck!
Annie,
It looks quite normal to me. Immortalized cell lines usually have higher proliferative activity than primary cultures, though my own experience mainly refers to neurons, where the difference is huge. Have you compared growth rate of your primary culture and this one? Our neuroblastoma cells make media yellowish after 48h of incubation.
Are you sure that induction problems are caused by pH stress?
Good luck!
Annie,
Have you found the answer? I have faced similar problem in case of differentiated 3T3L1. right from D3, medium was becoming lemon yellow and cells were peeling off. There was no bacterial or fungal contamination. When I shifted the cells to Gibco FBS, the problem was solved.
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