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Problems in MDAMB-231 growth - (Feb/15/2011 )

Hi,
I have been trying to revive MDAMB-231 using different vials frozen on different dates for over a month now without any success. The day after reviving, the cells look fine, thats when I change the media. The day after media change the cells die. I have used both DMEM with 10% serum, glutamax, antibiotics and also RPMI. The growth conditions are 37C, 5% CO2. I am sure the media is fine, because I have used them for other cell lines which are growing nicely.
I got another MDAMB-231 plate from a neighbouring lab and split and tried to grow them in my lab. But again, the cells are very unhealthy. Most of the cells are dying on the day aftr splitting and the few attached cells look unhealthy. Please Help.

-Sowjanya-

Do you pre-warming the medium before using? How long do you trypsinise for? How do you neutralise the trypsin?

-bob1-

Yes, i prewarm the media to 37C before using. Trypsinise for 5 mins. 7 mins maximum. and I neutralise the trypsin with the media.

-Sowjanya-

Ok, that looks fine, though you should only trypsinise until the cells are lifting off as seen under the microscope to prevent them clumping.

What medium were the cells in when you got them? If they were in a different medium to DMEM (or RPMI), you may have to wean them onto your medium.

-bob1-

No, they were not in different media. The cells frozen in DMEM were revived in DMEM and cells frozen in RPMI were revived in RPMI.
The confluency has been the same for over 2 weeks. Too many dead cells and the adhered cells are unhealthy. I am changing the media every alternate day.

-Sowjanya-

Hmmm, I really don't know what the problem is - all the clues point to a medium problem, but you said that you have tried the medium on other cells and they all grow fine.

Density problems - overconfluent when split? It doesn't sound like that should be a problem though.

-bob1-

No, it cant be a media problem. Like I said, all the other cells are perfectly fine.
And its not even getting a chance to be overconfluent. the cells are dying very fast and very few cells remain adhered to the bottom. It has been 40% confluency for over a week. Evryday I check, its the same confluency and increased no. of dead cells.

Its a complete mystery to everybody in the lab. The cells are just refusing to grow :(

-Sowjanya-