sudden cell death- Please HELP - (May/02/2008 )
Hi all
I'm really hoping someone can help me!
I have established 15 independently stably transfected cell pools (i.e. not clones) of NIH 3T3 cells with different constructs. I am splitting them before they reach 70% confluence and I grow them in 10% FCS, they look perfectly fine. However, I have also seeded all 15 for foci forming assays in 5% FCS. I have done this several times before and it always worked fine, the cells grow in a pretty monolayer except in the places where they form foci obviously. But this time something has gone very wrong. After 12 days at confluence some of the cell pools have started dying!, They simply de-attach!. I do not se any infection, no little black dots moving around. I also checked for mycoplasma using PCR and there is no infection. The medium has a nice red colour. Why are they dying?. What is worse is that the ones that are dying so far are ones transfected with control constructs i.e. constructs that I have used before for the exact same type of experiment where I did not have this annoying problem. So I don’t get it. What is wrong?!. Could it be some invisible infection that is only displayed under stress? (because they look fine in the flasks I split frequently). PLEASE HELP!
Jensen
Did you change _any_ batch recently? I mean medium, FBS, palsmid, flasks whatever? If the answer is yes, that should be the problematic ingredient.
Well, We have gotten new batches of FBS, flasks and medium from the same companies as usual. Yet I think if it was a problem with one of these things all my 15 pools would die during the confluent growth and not just some of them -or am I wrong?
jensen
This happened to us once. It turned out to be a CO2 problem. The CO2 sensor of the incubator was out of order so the CO2 concentration was wrong. Once we fixed the incubator, everything turned to normal.
That's also a good idea. But the high/low CO2 concentration can easily be detected by the colour change of the medium (pH). If the medium is buffered by HEPES or similar, then the pH should not change too much, and the cells should not be affected by the CO2 either. Unless I'm wrong :)
That's interessting. Did you see cell death with all the cells or only some?
We also had problems with the CO2. Almost all cells behaved different after too high / low CO2. My cells looked okay, but the didn't work well in the following experiments.
But if you got new FBS, I would check this first. As long as it is not FBS of the same lot, you will always have different composition.