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problem in MTT assay - (Aug/22/2012 )

Hi, Guys. I am brand new in cell culture research. Please help me!!!
I have a problem when I did MTT assay today.
I have added toxic reagent to the neurons. After 20hrs incubation, I use the Invitrogen MTT assay kit to see how many neurons survived.
After the MTT solution in PBS was added in the 96 well plate with neurons, it looked like normal, pretty yellow color. then I incubated them in the incubator for 4 hrs. Normally the solution in each well is still yellow color before the acidic solution is added. But this time I found some of the wells have become very dark blue. I looked at these wells using microscope, there was no neurons at all except a lot of blue color substance. I assume it is the metablized product from MTT. But if all of the neurons died, why there is still metablized product? If it not because of the neuron death, then why?

Thank you very much!!!!



Did you remove the old medium containing your test compound before adding the MTT containing medium? Though you got cell killing in those wells, the released enzymes remained active and could continue to convert MTT.

After you remove the medium, the less toxic wells (with more viable cells) should be bluer than the more toxic wells (with less viable cells). If you did remove the old medium, one remote possibility might be that the test compounds in those blue wells stimulated the expression or activity of the mitochondrial redox enzymes?

Why do you prefer MTT? Maybe you would like our AquaBluer. Similar to MTT, AquaBluer measures mitochondrial redox acivity, but it does not require cell lysis and DMSO dye solubilization. It is simpler to use; you just add-incubate-read. Your MTT kit may cost $194/kit for 1,000 assays in 96-well format, AquaBluer kit is $199/kit for 15,000 assays in 96-well format. That's 15x cheaper.


Does anyone used Vybrant® MTT Cell Proliferation Assay Kit (V-13154)?
is that ok if i used the quick protocol option which use DMSO rather than use SDS-HCL ?
i want to reduce the incubation time by using that ok?

can anybody help?
this is the product information.