Cells suddenly died after changing the medium... why? - (Oct/28/2009 )
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.
Did you change completely, or do a gradual shift to Optimem?
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?
lab rat on Oct 28 2009, 11:21 AM said:
"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?"
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.bdbiosciences.com/external_file.../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.
Thanks for the detailed information. Appreciate it!