reduced serum?? - questions to antibiotic concentration & time (Jul/01/2010 )

i am currently perfoming my first cytotoxicity/proliferation studies with my cells!

1. Q: in some publications they reduce FCS from 10% to 1% when they are performing their tests!! do i have to reduce my antibiotics also? - because it is known that some compounds in the serum bind to antibiotics (make them less toxic to the cell itself)! or should i completely avoid the use of antibiotics completely?

2. Q: do i also have to make some preliminary tests to evaluate how cells behave with 1%FCS? or is it ok to reduce serum for 48h?

thx in advance

Hi,

You could have prepared a stock of serum free medium with antibiotics. Then serum is to be added as required. That way you don't reduce the concentration of your antibiotics.

For proliferation assay, as i understand it, you subject the cells to low or no serum medium to render the cells quiescent at which point you would expect most if not all cells come to G0 point of the cell cycle. Some cells require longer than others in this regards, depends on the cell doubling time. When you then add your reagent to stimulate cell proliferation, they will all take off from the same point, G0, and hence in theory, giving you a more reliable end point result.

Some cells are more susceptible towards low or no serum medium in that they go into spontaneous apoptosis. You can consider adding some supplement if that is the case, such as ITS (insulin, transferrin, selenium) when you change the medium to low or no serum medium. This again, is cell type dependent.

If i were in your shoes, i will certainly try growing the cells in 1% FCS and see how long can the cells survive.


Best,