MTT assay overestimates human airway smooth muscle cell number in culture

In this study, we evaluated the accuracy of the MTT (a tetrazolium salt) assay for cell counting by comparing it to haemocytometer. When airway smooth muscle cells were cultured in RPMI with fetal calf serum < or = 5%, no significant differences were observed in cell number counted by the two methods. For cells cultured in 10% serum, however, the result was 20% higher when counted by MTT assay. Rhodamine 123 uptake was similar in ASMCs cultured in the presence of serum at 5 and 10% indicating no difference in total mitochondrial activity per cell. Serum at > 5% may modulate the activity of enzymes responsible for reduction of MTT. Our results indicate that the MTT assay may not be accurate under certain conditions, especially when the treatment of cells can affect the enzymes that account for MTT reduction in addition to stimulating cell growth.