Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons
- 作者: Surin A.1,2,3, Sharipov R.2, Krasil’nikova I.1, Boyarkin D.1,3, Lisina O.2,4, Gorbacheva L.3, Avetisyan A.5, Pinelis V.1
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隶属关系:
- Scientific Center of Children’s Health
- Institute of General Pathology and Pathophysiology
- Pirogov Russian National Research Medical University
- Moscow Technological University
- Belozersky Institute of Physico-Chemical Biology
- 期: 卷 82, 编号 6 (2017)
- 页面: 737-749
- 栏目: Review
- URL: https://journals.rcsi.science/0006-2979/article/view/151420
- DOI: https://doi.org/10.1134/S0006297917060104
- ID: 151420
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详细
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 (ΔΨm), 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 ΔΨm-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 ΔΨm 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.
作者简介
A. Surin
Scientific Center of Children’s Health; Institute of General Pathology and Pathophysiology; Pirogov Russian National Research Medical University
编辑信件的主要联系方式.
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 119991; Moscow, 125315; Moscow, 117997
R. Sharipov
Institute of General Pathology and Pathophysiology
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 125315
I. Krasil’nikova
Scientific Center of Children’s Health
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 119991
D. Boyarkin
Scientific Center of Children’s Health; Pirogov Russian National Research Medical University
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 119991; Moscow, 117997
O. Lisina
Institute of General Pathology and Pathophysiology; Moscow Technological University
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 125315; Moscow, 119454
L. Gorbacheva
Pirogov Russian National Research Medical University
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 117997
A. Avetisyan
Belozersky Institute of Physico-Chemical Biology
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 119991
V. Pinelis
Scientific Center of Children’s Health
Email: surin_am@mail.ru
俄罗斯联邦, Moscow, 119991