formazan upon reduction.
The process primarily takes place in the cytoplasm, and to a
lesser extent in the mitochondria and cell membrane (5–7). The
reductase activity in the ER is highly dependent on the concentration
of intracellular NADH and NADPH. The abundance of
these nucleotide cofactors is associated with the availability of
extracellular glucose. Hence, exhausted cell culture medium may
cause lower MTT absorbance readings due to its low glucose
concentration. The mitochondrial succinate dehydrogenase and
cytochrome c take part in MTT reduction as well (8). Therefore,
any substance or treatment that interferes with these enzymes or
with glycolysis, changes the rate of MTT reduction, and consequently
alters the result of cell counting.