Surin A M, Sharipov R R, Krasil'nikova I A, Boyarkin D P, Lisina O Yu, Gorbacheva L R, Avetisyan A V, Pinelis V G
Scientific Center of Children's Health, Ministry of Healthcare of the Russian Federation, Moscow, 119991, Russia.
Biochemistry (Mosc). 2017 Jun;82(6):737-749. doi: 10.1134/S0006297917060104.
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), 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-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 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.
基于将细胞质中的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑还原为具有强烈吸光性的甲臜的MTT法，是测定细胞活力和NAD依赖性氧化还原酶活性最常用的方法之一。在本研究中，研究了MTT（0.1mg/ml）对培养的大鼠小脑神经元和分离的大鼠肝线粒体的线粒体膜电位（ΔΨ）、细胞内NADH和呼吸作用的影响。MTT导致NADH自发荧光、MitoTracker Green（MTG）荧光以及ΔΨ敏感探针Rh123（罗丹明123）和TMRM（四甲基罗丹明甲酯）的荧光迅速淬灭。与NADH、MTG和TMRM不同，Rh123信号在5-10分钟后在核质中增加，同时伴随着细胞质和神经突中不透明甲臜聚集体的形成。Rh123信号的增加表明由于ΔΨ降低，探针从线粒体扩散到细胞质和细胞核。呼吸链复合体I的抑制降低了甲臜形成的速率，而复合体IV的抑制则增加了该速率。复合体III和ATP合酶的抑制对甲臜形成速率的影响微不足道。2-脱氧-D-葡萄糖对糖酵解的抑制阻断了MTT的还原，而丙酮酸则以浓度依赖的方式增加了甲臜形成的速率。MTT将培养神经元的耗氧率降低到呼吸链复合体I和III同时被阻断时观察到的值，并且如果使用由NAD依赖性脱氢酶氧化的底物，它会抑制分离线粒体的呼吸作用。这些结果表明，培养的大鼠小脑神经元中甲臜的形成主要发生在线粒体中。甲臜形成的初始速率可作为复合体I活性和丙酮酸转运速率的指标。
