I have no experience with MTT in hypoxia but given the mechanism it shouldn't be a problem. If I recall correctly MTT acts as an electron acceptor from mitochondrial dehydrogenases (presumably ETC) and therefore oxygen shouldn't be needed. But I guess the "accuracy" depends on what you're really trying to measure - toxicity of some substance, cell number,...? Comparing MTT signal in hypoxia with normoxic (or rather hyperoxic) cells might be misleading.

Thanks for your answer. I am comparing normoxic vs hypoxic viability so would have initially thought that MTT would be fine but just wondering whether the reaction of formazan reduction would occur in low oxygen or whether the reduction reaction is primarily based on mitochondrial oxygen donation.

The MTT assay should work as long as you are using a hypoxia chamber to induce the hypoxia and not an antioxidant or reducing agent. These agents will almost certainly directly reduce the MTT giving you false high readings. The assay will make it appear that all the treated cells are growing very rapidly even if they are dead. If you are doing the assay in the hypoxia chamber it may require more time to develop. However since you will most likely need to remove the cells from the chamber in order to add the MTT, the cells will end up being exposed to normoxic conditions. In any case, if you want to compare the effectiveness of treatments under each condition, be sure to include all the controls on each plate and compare the percentage change rather than the ODs. It may be simpler to just do the assay under normoxic conditions since it is a fairly short end point assay (that is after treating the cells under hypoxic conditions for a day or more, the few hours for the assay is unlikely to significantly change the cell number).

Ramyar Chavoshinejad Sir,You said MTT for mitochondria activity I was performing with isolated mitochondrial samples what is the time required for incubation after adding MTT.Does it vary with the source of mitochondria.

Thank you all for your answers. They have been very helpful.

Thank you sir