MTT assay incubation time - general MTT questions (Mar/23/2010 )

I have been trying to do the MTT assay to detect apoptosis in my cancer cell line. The protocol I have says to incubate for 1 to 4 hours. How do I determine what the optimal incubation time is?


My current procedure:
Plate cells in 96 well at a density of ~5000 cell well.
Incubate overnight for 12 hours or for 72 hours.
Add 10ul of MTT reagent to each well.
Incubate for 2 hours.
Measure OD at 490nm.
Compare readings at 12 hours and 72 hours.


My PI says that the changes I am observing in OD are not large enough to be significant. But I see changes in cell number when I look at the cells under the microscope. Thus, I believe I am doing something wrong with the assay. I have been wondering about incubation time. Should I talk to the company the manufactures the MTT reagent I use?

Any other helpful MTT tips or links to read up on the assay are more than welcome. I really am a "newbie" in the lab.

PLEASE HELP!

Do you see a colour change?
I use the MTS reagent (from Promega - similar to MTT) and I can see a colour change in my cells which increases with intensity over time.
I think one of the advantages of MTS over MTT is that you don't have to stop the reaction to read your plate - you can read at 1 hour and then put back in the incubator to read again at 2, 3 or 4 hours.
Other than that, I'm sorry I can't be more help.