how did it happen? - cells grow normally but when in SFM media, things get worse (Nov/14/2005 )
My culture grew normally in DMEM+10% fetal calf serum, whereas when i changed the media to serum free media, the situation became very different:the culture media became turbid and then the attached cells began to slough. At first, i doubt the serum free media was contaminated.Then i use media alone culture-without cells as a control, after sereral days ,there was no signs of contamination.Therefore it seemed the media was ok and the cells were suspious of contamination. Then i sample the supernatant fluid (of cells culture in DMEM 10% fetal calf serum) and under microscope i saw traces of bacteria,which caused the whole problem.
But i still don't understand why in FCS media,cells grow normally but when in SFM media, things get worse? AND is it still Ok i culture cells in FCS media where the contaminated bacteria seemed to cause little problem?
SFM arrests the cell in the g0/g1 phase. sometimes it is used to synchronize cells. However, if used for more than 12-16 h you willl definitely see cells losing their adherence and floating to the top.
But i still don't understand why in FCS media,cells grow normally but when in SFM media, things get worse? AND is it still Ok i culture cells in FCS media where the contaminated bacteria seemed to cause little problem?
After 12-24. your cells will undergo apoptosis in SFM.
First,i want to thank you for your kind replies.
And sorry i didnt make it clear. When i said serum free medium, i didnt mean basic media (DMEM) without adding FCS. i meant comercialized SFM ,which can be used as alternative for Basic media with FCS.
And sorry i didnt make it clear. When i said serum free medium, i didnt mean basic media (DMEM) without adding FCS. i meant comercialized SFM ,which can be used as alternative for Basic media with FCS.
When you change medium from DMEM+FCS to SFM, you may need to do adaptation.
1. Check that your cell has >90% viability.
2. 75% DMEM+FCS and 25% SFM of the total volume of medium
3. 50% DMEM+FCS and 50% SFM
4. 25% DMEM+FCS and 75% SFM
5. no DMEM+FCS and 100% SFM
6. continue growing them in 100% SFM
You may wish to freeze them in liquid nitrigen.
1. harvest your cell in mid-log phase of growth with viability >90%
2. 50% fresh medium and 50% used medium and 7.5% DMSO.
3. Cell density 0.5-1.0 x10^7 cells/ml
I hope this may help.
I feel that subtitle of my post is not quite clear and misleading.
Maybe i'd better put it ito this:"sligtly contanminated cells grow normally but when in SFM media, things get worse." What really puzzles me is that the contaminated bacteria seem prefer SFM and inhibited by DMEM+FBS media?
But thanks anyway,Minnie Mouse 
Maybe i'd better put it ito this:"sligtly contanminated cells grow normally but when in SFM media, things get worse." What really puzzles me is that the contaminated bacteria seem prefer SFM and inhibited by DMEM+FBS media?
But thanks anyway,Minnie Mouse
Sorry for the misunderstanding. Did you put penicillin in SFM?
Yes,penicilin and streptomycin. In fact, five fold of nomal administration didn't help much controlling bacteria growth in SFM cultivation.
There is a possibility that SFM is richer compared to 10% FCS. Since your bacterial population is taking all nutrients your cells are dying. If you have high bacterial contamination pen/strep will not help, it will reduce but will not clear it. Start with completely new batch of cells(do not use frozen stock from same batch) and try.
Just one more question. Does your cells need any extra growth factors that are not included in SFM. Also you should check with your supplier of SFM, that PEM/strp is ok with taht media and if SFM causes some turbidity(some times SFM does develop turbodity)?
aside from the fact that it appears your SFM makes your bacteria very happy, it's also true that serum contains many active immunological factors
perhaps this is why your bacteria do not grow so well in the DMEM/FCS?