we have had problems with J774A.1 for over a year now and we don't now what else to try. They seem to grow fine in flasks but when we plate them into plates to do transfections, every now and then, randomly, they will die. One day after plating, even before transfecting, cells in the wells will be devastated, specially in the middle of the well. This always seems to happen after passage into plates, but not every time we do it. Also, it doesn't happen in 100% of the wells, but in most of them. We can go for weeks no problem, and then, one day, for no apparent reason, the "effect" will happen again.
Things we have noted and tried/checked with no sucess:
- our media is never older than a week
- our cells are always low passage (mainly because everytime this "effect" occurs we thaw a new vial and start from scratch
- we are carefull with trypsinization time and we always trypsinize+scrape to detach cells as they are so highly adherent
- we have tried different plastics
- we have tried different CO2
- we have tried culturing the cells in 20% FCS instead of the usual 10%
- we have ordered new vials from ATCC
This is tremendously inconvenient as we plan weeks of experiments but we can never be sure if the cells will do ok or if all the plates we just prepared will be wiped out the next morning.
I would deeply appreciate any comments or suggestions or to hear of anyone who might have had a similar problem and was successful solving it.
Also, does anyone have experience with any of the other J774 cell lines? I found another thread asking about these other cell lines but not much discussion followed so I wonder if anyone has more current knowledge about the issue.