Нажмите на эту строку чтобы перейти к Новостям сайта "Русский врач"

Перейти
на сайт
журнала
"Врач"
Перейти на сайт журнала "Медицинская сестра"
Перейти на сайт журнала "Фармация"
Перейти на сайт журнала "Молекулярная медицина"
Перейти на сайт журнала "Вопросы биологической, медицинской и фармацевтической химии"
Журнал включен в российские и международные библиотечные и реферативные базы данных

ВАК (Россия)
РИНЦ (Россия)
Эко-Вектор (Россия)

THE STUDY OF THE YELLOW TETRAZOLIUM SALT REDUCTION TO FORMAZAN IN HеLa CELLS

DOI: https://doi.org/10.29296/25877313-2023-11-08
Issue: 
11
Year: 
2023

A.Yu. Semushkina
Post-graduate Student, Research Scientist, Laboratory of Atomic and Molecular Bioregulation and Selection,
All-Russian Scientific Research Institute of Medicinal and Aromatic Plants (Moscow, Russia)
E-mail: syomushkina@vilarnii.ru
D.S. Kabanov
Ph.D. (Biol.), Leading Scientist, Biomedical Technology Department,
All-Russian Scientific Research Institute of Medicinal and Aromatic Plants (Moscow, Russia)

Relevance. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test is one of the most common used methods for determining cell viability (cytotoxicity) during cell response to plant’s extract or individual extract’s compound. Despite of extensive use of this method it remains unclear whether plasma membrane of cells is destroyed by MTT-formazan crystals during MTT reduction to MTT-formazan; whether the MTT-formazan is lost after cell destruction by MTT-formazan crystals. The purpose of this study was to assess by microscopy and spectrophotometry methods the possibility of MTT-formazan releasing into cultural medium during MTT reduction to MTT-formazan by HeLa cells. Material and methods. HeLa cells were grown on coverslips in Weighing bottles, incubated with MTT (0.1 or 0.5 mg/ml final concentrations) and visualized by light microscopy after 30 min, 60 min, 120 min and 240 min after MTT addition. For spectrophotometry measurements cells (5×104 cells/well) were added to a 96-well plate followed by MTT. The absorbance spectrum was recorded after 30 min, 60 min, 120 min and 240 min after MTT addition. Results. Microscopic examination revealed two phases in MTT reduction by cells to MTT-formazan – the formation of granules and crystals of MTT-formazan. The maximum ratio of crystals to granules of MTT-formazan and almost complete destruction of cells were observed by light microscopy at 240 min of incubation with MTT. However, in the study by spectrophotometry after cell destruction by dimethyl sulfoxide the values of optical density of the MTT-formazan after 120 min as well as 240 min of incubation were almost the same. Therefore, the transformation of MTT-formazan from granules to crystals does not affect the optical density despite of cell destruction. Culture medium without cells did not reduce MTT to MTT-formazan. In the supernatant from cells without their destruction the significant production of MTT-formazan was also not observed. Conclusions. MTT-formazan crystals do not dissolve in the culture medium but dissolve in hydrophobic solvents such as dimethyl sulfoxide. In this regard the destruction of cells by MTT-formazan crystals does not lead to significant loss of MTT-formazan during MTT reduction by HeLa cells and does not significantly effect on results of cytotoxicity determined by MTT test.

Keywords: 
HeLa
МТТ
Formazan
MTT-formazan granules
MTT-formazan crystals
light microscopy
spectrophotometry.

References: 
  1. Braissant O., Astasov-Frauenhoffer M., Waltimo T., Bonkat G. A review of methods to determine viability, vitality, and meta-bolic rates in microbiology. Frontiers in Microbiology. 2020; 11: 547458.
  2. Riss T., Moravecs R., Niles A., Duellman S., Benink H., Wor-zella T., Minor L. Cell viability assays. Assay Guidance Manual [Internet]. 2016; 1–25.
  3. Ghasemi M., Turnbull T., Sebastian S., Kempson I. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. International journal of molecular sciences. 2021; 22 (23): 12827.
  4. Stockert J.C.; Horobin R.W.; Colombo L.L.; Blázquez-Castro A. Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018; 120: 159–167.
  5. Bernas T., Dobrucki J. The Role of Plasma Membrane in Bioreduction of Two Tetrazolium Salts, MTT, and CTC Arch. Biochem. Biophys. 2000; 380: 108–116.
  6. Bernas T., Dobrucki J.W. Backscattered light confocal imaging of intracellular MTT‐formazan crystals. Microscopy research and technique. 2004; 64 (2): 126–134.
  7. Fisichella M., Dabboue H., Bhattacharyya S., Saboungi M. L., Salvetat J. P., Hevor T., Guerin M. Mesoporous silica nanopar-ticles enhance MTT formazan exocytosis in HeLa cells and astrocytes. Toxicology in vitro. 2009; 23 (4): 697–703.
  8. Molinari B.L., Tasat D.R., Palmieri M.A., Cabrini R.L. Kinetics of MTT-formazan exocytosis in phagocytic and non-phagocytic cells. Micron. 2005; 36 (2): 177–183.
  9. Lü L., Zhang L., Wai M. S. M., Yew D. T. W., Xu J. Exo-cytosis of MTT formazan could exacerbate cell injury. Toxicology in vitro. 2012; 26 (4): 636–644.
  10. Liu Y., Peterson D. A., Schubert D. Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis. Proceedings of the National Academy of Sciences. 1998; 22: 13266–13271.