MTT assay is one of the widespread methods of determining the viability of cells in culture with different pharmacological treatment. Intracellular dehydrogenases reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) to formazan, which absorbs light in the spectral region <600nm. Formazan forms aggregates in the cells which appeared as dark spots. In the present work, we performed a light microscopy study on the impact of MTT on mitochondrial potential (ΔΨm) and the light absorption by cultured neurons from rat cerebellum. Changes of ΔΨm were recorded employing fluorescent potential-sensitive probe rhodamine123 (Rh123). Application of MTT (0.1mM) caused a rapid and almost complete quenching of fluorescence Rh123, followed by (in 5-10 min) fluorescence increase in the nucleoplasm. Rh123 diffused out of the mitochondria to the cytosol followed by diffusion into the nucleus, which is a characteristic feature of the ΔΨm fall. Equally rapid has been quenching of the MitoTrecker Green (MTG) fluorescence, however without subsequent fluorescence increase, because MTG irreversibly binds to mitochondria. Growth of Rh123 fluorescence coincided with the beginning of absorbtion of light passing through the cells, due to formation of formazan. MTT also caused irreversible reduction in the neuronal NADH autofluorescence. Rising phase of the Rh123 signal and decrease of the cell culture light transmittance likely reflects restriction of NADH oxidation by respiratory chain complex 1 because NADH is spent on the restoration of MTT to formazan. As a result, operation of the complex 1 is ceased and mitochondrial depolarization develops. The results obtained suggest that the MTT (at concentrations of 0.1 mM and above) causes rapid mitochondrial dysfunction and therefore should be treated with caution in the interpretation of data concerning cell survival and dehydrogenase activity based on the amount of formazan formed during the incubation of cell cultures with MTT.
neurons, mitochondrial potential, fluorescence microscopy, MTT
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