How much trypsin/Cause of death - (Dec/15/2010 )

I'm not sure it was over growth or a bacterial infection that killed my A459 cells, but they're all dead. The media turned a shade that was slightly purple-ish (DMEM, 10% FBS, 1% strep). All of the cells in the flasks died, as well as 3 of the 4 96 well plates that I had plated yesterday, and the cells looked perfectly normal yesterday. So I'm trying to rule out all possible causes for their death.

Based on the color of the media, and the fact that all other cell flaks in the incubator survived without problems (CO2 didn't run out etc), I'm thinking possible over growth, but it seems very unlikely based on the number of cells I had yesterday.

Is 1% strep enough? Or should I modify it? In the past 1% has done the job without issue...?

Bacterial infection? Under the microscope there are a few long stringy (very scientific wording I know) items in there with the dead and detached cells. The morphology of the dead cells looks fairly normal, a little different but not much, except that they aren't attached to the bottom of the flask, but are in large "rafts" so to speak.

Possibly too rough centrifuge? The centrifuge I always used in the past is broken, so I used a different centrifuge. I normally spin them at 1500 rpms for 5 mins, this time I spun them at 1100 rpms for 8 mins. (1100 rpms for 5 minutes left a large amount of the cells still in suspension so I did it for another 3 minutes) But this is unlikely, since I have 1 plate that survived and none of the others survived.

Myo tested negative.

Whatever it was that killed the cells, I have one 96 well plate that survived, so I want to transfer the cells in the wells in this plate back into a culture flask. How much trypsin should I add, just enough to cover the bottom of the wells? When doing subculturing I use 5mL in the culture flask.


Thank you very much for your help. I know I have a lot of silly questions, but please bear with me (and talk slowly) as I am a chemist by training and have never done any cell culture work in the past. Baptism by fire?

Purple = high pH = bacterial infection normally, unless you happen to have added base to your medium...

Well, I didn't add any base to my media that I know of. FBS and strep/pen aren't basic, and I went ahead and verified this with a tritration yesterday. I titrated the media that I had used, FBS, strep/pen, as well as original media, and the media from the dead cells. The media from the dead cell flask had a pH of 9.7 and the original media that was put in had a pH of 7.9. Are these the numbers that one would expect to find? (I only titrated each sample once, so I don't have the proper stats for these.)

The cells that were alive in the 96 well plate were successfully transfered back to a new culture flash. When I left last night at 5pm they looked to be in good health and had adhered to the flask. When I got in today, they were all dead and floating, and once again the media was a hue of purple.

So I had no choice today but to thaw out a cryovial. I mixed up NEW media, in order to rule out the possibility that the media itself was what causing the problem/contaminated. I'll report back with the results of this tomorrow.

Any ideas as to what could have caused them to become contaminated in the first place, and why cells that were healthy the day before would all be dead and the media so basic? Our stock of these cells is very small, only 2 more cryovials remain. I have been freezing down 2 or 3 vials worth per week, and this problem hit me before I could build up a substantial enough stock, so I really can't afford to kill the few remaining cells.

Thanks for all your help!

I suspect bacteria purely because the high pH is a common indicator of bacterial infection (though I don't know which bacteria). If this is a very precious cell line (as in "you can't buy/get it from somewhere else") you could treat with stronger antibiotics and use multiple antibiotics such as gentamycin, penicillin, neomycin, but this is not advised as it can damage/change how the cells behave.