Related Experiment Video
Updated: Oct 8, 2026
Live-imaging of Mitochondrial System in Cultured Astrocytes
Published on: November 16, 2021
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological
I Isobe1, K Yanagisawa, M Michikawa
1Department of Dementia Research, National Institute for Longevity Sciences, Nagoya City University Medical School, Japan. iisobe@med.nagoya-cu.ac.jp
Abstract:
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.
Insights
The widely used 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay can alter cell behavior, impacting viability measurements. MTT affects cellular compartments and Akt phosphorylation, potentially influencing assay results.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a common reagent for assessing cell viability and cytotoxicity.
- The intrinsic biological effects of MTT on cellular functions remain largely uninvestigated.
Purpose of the Study:
- To investigate the direct cellular effects of MTT beyond its role in viability assays.
- To determine if MTT influences intracellular pathways relevant to cell signaling and function.
Main Methods:
- Cultured rat astrocytes were treated with MTT.
- Immunofluorescence staining using anti-Rab5 antibody was performed to assess morphological changes in endosomal compartments.
- Western blotting was used to analyze the dissociation of early endosomal auto-antigen (EEA1) and the phosphorylation status of Akt.
- The involvement of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway was inferred.
Main Results:
- MTT induced morphological alterations in an intracellular membranous compartment identified by Rab5.
- Dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction was observed.
- Phosphorylation of Akt was detected, suggesting activation of the PI(3)K pathway.
Conclusions:
- MTT exerts direct biological effects on cultured rat astrocytes, influencing cellular compartments and signaling pathways.
- These MTT-induced cellular changes may introduce discrepancies in cell viability measurements obtained through MTT assays compared to other methods.
- MTT and related tetrazolium salts show potential as tools for studying Akt and PI(3)K pathway activation mechanisms.
