2017/06/01 by A. M. Surin, Alexander Surin, Р. Р. Шарипов +12 · 1 citation
Biochemistry, Genetics and Molecular Biology · Chemistry · Neuroscience · #Biochemistry #Biology #Biophysics #Cell #Cell growth #Cellular respiration #Chemistry #Citrate synthase #Cytosol #Enzyme #Formazan #Intracellular #MTT assay #Mitochondrion #Molecular biology #NAD+ kinase #Neuroscience and Neuropharmacology Research #Photoreceptor and optogenetics research #Respiratory chain #Rhodamine 123 #Viability assay #bioluminescence and chemiluminescence research
paper · doi:10.1134/s0006297917060104
crossref issued 2017/06/01 · crossref published 2017/06/01 · crossref published-print 2017/06/01 · openalex publication_date 2017/06/01 · crossref created 2017/06/14 · crossref published-online 2017/06/15 · openalex created_date 2025/10/10 · crossref deposited 2026/03/16 · crossref indexed 2026/07/25 · openalex updated_date 2026/07/31
The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ m ), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ m -sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.