Serum concentration in treatment of cells for MTT assay - (Dec/11/2010 )

Hello everyone,

I will be evaluating the anti-cancer activity of several crude extracts on breast cancer cells using the MTT assay. My question is, should the serum content of the media be reduced when i treat the wells with my extract? I have come across lab members who reduce the serum content of their wells during treatment while others keep it at 10% supplemented media. Any feedback would be most welcomed

My proposed protocol is as follows:
Day 1
Seed cells at 6000 cells /well suspended in 10% supplemented media in 69 well plates.

Day 2
add extracts at concentrations of 0, 15, 25, 50, 75, 100 microgram/ml
* Should serum free media be used here? or maintain at 10% supplemented media?

incubate for 72 hours.

Day 4.
add MTT, incubate for 4 hours, dissolve formazan with DMSO and read plate at 570nm.

Unless you have a normal growth control (i.e. the %FBS that you use for growing the cells normally) comparing reduced serum results with normal growth is impossible. I would keep the serum at the same concentration as you use for normal growth.

Sometimes serum does interfere with the action of drugs, in these cases you can use reduced serum, but you MUST include proper controls.