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MTT assay is not working - (Oct/01/2009 )

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I have had the same exact problem and I havent been able to figure out why I can't get the MTT assay to work. I am following the protocol given by invitrogen. If you figured it out let me know.


I have encountered the same problem, after plating 48 hours, all the cells died. I can confirm that it shouldn't be caused by something in MTT. If you solved this problem, please tell me. Thank u very much.


Hi slyphe,

I personally do not have any experience in culturing fibroblast culture. However, I did a lot of MTT on primary culture and continous cell line.

May I know is the fibroblast culture of yours is actually a primary culture?

This is what i normally do for MTT assay particularly when involving primary culture or any new cell line that I have not work with previously :
1. determine the plating efficiency, by plating a 96 wells plate with the cell of interest with different cell concentrations. this is important as certain cell can't grow well when they are been plated in small number in a 96 wells plate under certain cell concentration.
2. let the cells to grow further for another 72 till 96 hours to ensure that they can actually grow well in a 96 wells plate. this would be helpful as well to determine which cell seeding density which actually gives you the over confluent cell culture in a 96 wells plate.
3. after determine the plating efficiency, then optimize the cell seeding density which would give you the OD around 1.5 at the end of your experiment.

Oh ya, before you do any seeding or experiment, please ensure that you determine the cell viability, at least via tryphan blue staining, while you do the cell counting. Make sure you only proceed further with your experiment when the cell viability is at least 90% viable. In my experience, i get a very reliable and consistent and reproducible results accross 100 96-wells plate whenever my cell viability exceed 95% just before i plate the cells.

In cases that the fibroblast cells can't actually grow well in a 96 wells plate, perhaps you should do some optimization in determining the best cell culture medium and condition for your cells.

And I hardly have the time to actually removing the MTT via pipette, but just merely flip the plate over to remove the MTT if it is a anchorage-dependent cells, as I am processing 20 to 100 plates each day. And it does perfectly in my hands.

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