what's wrong with my MTT assay on a suspension cell line - (Dec/07/2007 )

recently I am testing the cancer cytoxity of the PS-341 with MTT assay on the myeloma cell lines, named RMPI 8226. however, I found that the formation of formazan salt is not ok at all. after 4 hours of incubation with the MTT, seldom cells contain formazan crystals. the outside rows including the control group and the highest drug-concentration group showed some formazan formation, but the middle rows show nothing but the yellow cells!! and the cells seem have died. I just don't know what is the possible problems with my MTT assay.
my MTT protocol is as following:
seeding 100ul cell suspension with 5000cells/well in a 96-well plate, and at the same time add the drugs
48 hours later, I found that the cells become adherent to the wells instead of suspension status, and the proliferation is not very obvious even in the control row
add 10ul MTT at 5mg/ml into each well and inocubate for 4 hours. the MTT was prepared and stored at -20C in dark and thawed just before use.
add SDS/HCL to solve the crystal.
my cells grows quite well in the flasks but not in the wells.

With an MTT assay, if the wells are yellow, then the cells have died. Others may have more advice, but it seems that your problem probably is with your cell growth, and not the MTT assay itself.

Have you tried preincubating the cells in the wells before adding the drugs? Also, I know that the MTT kit from ATCC has instructions on how to best decide the concentration of cells and length of incubation to get useful results. Just going by what you've said already, though, it sounds like you may need to extend the time you incubate the cells in the wells before adding the MTT.

thanks thesquire.
in fact, I have tried recenttly to add MTT directly into the non-treated cells and observe the cells under microscopy just after the addition of MTT without incubation. I found that the cells died instantly when the MTT was added. the cells are yellow and the shape became irregular and the membrance shrinked!! of course, after 4 hours incubation, the cells showed no crystal formation.
so, I just wonder if the MTT is toxic to some specific cells. if the MTT will kill my cells instantly, then the cells can't live long enough to allow the MTT come into the cells and transformed into formazan crystal. I think my situation is quite strange. so I need more advices from you guys. thanks a lot

thanks thesquire.
in fact, I have tried recenttly to add MTT directly into the non-treated cells and observe the cells under microscopy just after the addition of MTT without incubation. I found that the cells died instantly when the MTT was added. the cells are yellow and the shape became irregular and the membrance shrinked!! of course, after 4 hours incubation, the cells showed no crystal formation.
so, I just wonder if the MTT is toxic to some specific cells. if the MTT will kill my cells instantly, then the cells can't live long enough to allow the MTT come into the cells and transformed into formazan crystal. I think my situation is quite strange. so I need more advices from you guys. thanks a lot

I never add cold MTT to cells. Could it be a termic (not sure about the spelling) shock?
How do you prepare MTT? We have a powder we dissolve in PBS. We store the solution at 4°C (in dark) for maximum one month, but we prefer preparing the exact amount we need for the experiment.
We work with other cell lines but we do the same incubation time and way of formazan dissolution.

My lab uses the kit from ATCC, which has the MTT reagent already made up. I have to echo panda on this - if the cells are reacting that badly to adding MTT, perhaps it's the reagent solution you're using that's the problem. Do you make sure the MTT is warmed back up to room temperature (at least, if not warmer) before you use it? Are you sure you're not adding something bad when you make the reagent solution?

thanks everybody.
I use a kit too. the MTT reagent has been prepared and aliquoted and stored at -20C in dark. the reagent is thawed before use. I guess the problem is that I didn't warm up the MTT enough. I will try recently again.

hi,guys. I have tried the MTT again recently. after being warmed up in the incubator for about half an hour, my MTT work at last. the formazan forms well in the cells! so thanks everyone. and this lesson again tells me that the details determine everything.

hi i am also facing the same problem with MTT assay
i am not getting the gradation in OD from higher conc of drug to lower concentration of drug
could u pls help me

thanks and regards