Cells suddenly died after changing the medium... why? (View forum version)



ggshi00

Posted 28 October 2009 - 09:06 AM

In recent two weeks, when I change the medium of RPMI1640 10%FBS to OptiMEM I, the cells (HeLa cultured in 12-well plate) suddenly died...
The HeLa cells are good in flask. The cells can well attach to the plate and differentiate in RPMI1640 10%FBS. When I change the medium to OptiMEM I, the cells in Some wells suddenly died, but Others remain good. I never had such problem before, but these two weeks are killing me.
Any thoughts?
Many thanks!

lab rat

Posted 28 October 2009 - 10:21 AM

Did you change completely, or do a gradual shift to Optimem?

ggshi00

Posted 28 October 2009 - 10:29 AM

Completely. Remove all the RPMI1640 10%FBS, and then replace with Optimem.
It worked well before. One point I am thinking is: the RPMI1640 doesn't contain folic acid (for research purpose) and the cells have been passaged for over 20 times under the condition, will this be a problem?
Thanks!


Did you change completely, or do a gradual shift to Optimem?


lab rat

Posted 28 October 2009 - 01:15 PM

"One point I am thinking is: the RPMI1640 doesn't contain folic acid (for research purpose) and the cells have been passaged for over 20 times under the condition, will this be a problem?"

Could be.

I usually do a gradual shift to a new medium, over several changes. I wean hybridomas off IMDM +10% FBS onto a animal component-free, serum-free medium. I take several changes over a period of ~two weeks to accomplish this.

Once I have the hybridomas comfortably in a maintenance medium (100% IMDM + 5% FBS), I begin the weaning period by changing out 50% of the medium at a time. Each cell line has its own preference, so you will have to determine your own schedule empirically.

Here's the link to the procedure I use. http://www.bdbioscie.../353137_pug.pdf

Edited to add this info: I just read the Optimem insert. It says "for *most* applications no weaning is required to achieve 50% reduction in serum supplementation." Your cells may be an exception.

regards,
lab rat

ggshi00

Posted 28 October 2009 - 05:07 PM

Thanks for the detailed information. Appreciate it!