I'm culturing primary vascular smooth muscle cells. The protocol I'm following says to wean them from 20% to 10% FBS after passage 3. Can the reduction of FBS by 1/2 be done in one step or should I slowly reduce the amount of serum over several days?
Hello Lauren,
I think that Ramyar has a good point.
You dont want to drop the FBS concentration too fast because it might cause some unnecessary stress to the cells.
Iraklis
Gradual reduction is better for cell viability. To reduce from 20 to 10%, I would passage the cells and grow in 15% at least twice, then with next passage grow them in 10%. I think you will see less stress on the cells with a procedure like this.
Gradual reduction is a good general rule. However, there are some cell lines which are not under unduly stress if the concentration of the serum drops rapidly. E.g., in my experience, for embryonic stem cells such a drop usually induced differentiation and for melanoma B16F10 cells the drop from 10% FBS to 1% or even 0% is not problematic (0% FBS for 2 days and they are usually still feeling well and not in a big stress as analysis of mitochondrial activity shows). Which cell lines are which You got by empirical analysis - put the cells on two plates and check both changes in FBS, gradual and rapid, then check cell morphology, amount of dead cells (using, e.g. simple trypan blue) and/or WB for some stress proteins and/or analysis of mitochondrial activity.
My experience is that gradual is generally better (and many cases critical) but may not make much difference in the higher ranges. 10% serum is still a lot of serum for most cell types. Where gradual is generally critical is in the reduction to 1% or lower .