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Reduction of MTT by flavonoids in the absence of cells
Liang Peng1, Bochu Wang, Peng Ren
1Key Lab of Biomechanics and Tissue Engineering under the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, PR China.
Abstract:
The tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is used to determine cell viability in assays of cell proliferation and cytotoxicity. MTT is reduced in metabolically active cells to yield an insoluble purple formazan product. However, in this study, using a colorimetric method with MTT, we demonstrated that luteolin and quercetin (both are flavonoids) can reduce MTT in the absence of living cells. We also examined effects of some experimental conditions on the reaction, such as concentrations of flavonoids, incubation time, the structure and the existing form in solvents of flavonoids. The maximal absorbance values, which were 0.355 (luteolin) and 0.491 (quercetin), were observed when the concentrations of flavonoids were 200 microg/ml. Incubating MTT with flavonoids for 8 h, the absorbance values got the maximum, which were 0.320 (luteolin) and 0.398 (quercetin). The ability of flavonoids reducing MTT in RPMI-1640 with 10% fetal calf serum was higher than that in anhydrous ethanol. And the ability of quercetin reducing MTT was higher than that of luteolin both in RPMI-1640 with 10% fetal calf serum and anhydrous ethanol. Based on the results, this undescribed reaction can significantly influence the results of experiments using the MTT assay to measure the effects of flavonoids on cell growth.
Insights
Flavonoids like luteolin and quercetin can chemically reduce MTT assays, independent of cell viability. This non-cellular reaction impacts studies on flavonoid effects on cell growth.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The MTT assay is a standard method for assessing cell viability and proliferation.
- Tetrazolium salts like MTT are reduced by metabolically active cells to formazan, a colored product.
- Flavonoids are plant-derived compounds with various biological activities.
Purpose of the Study:
- To investigate the potential for flavonoids to directly reduce MTT.
- To characterize the conditions influencing this non-cellular MTT reduction by flavonoids.
- To assess the implications of this reaction for MTT assay results in flavonoid research.
Main Methods:
- Colorimetric MTT assay.
- Spectrophotometric analysis of formazan product.
- Testing varying concentrations of luteolin and quercetin.
- Evaluating effects of incubation time and solvent conditions (RPMI-1640 with 10% fetal calf serum vs. anhydrous ethanol).
Main Results:
- Luteolin and quercetin directly reduced MTT in the absence of cells.
- Maximal absorbance was observed at 200 microg/ml flavonoid concentration and 8-hour incubation.
- The reduction of MTT by flavonoids was more pronounced in RPMI-1640 with 10% fetal calf serum compared to anhydrous ethanol.
- Quercetin exhibited a stronger MTT-reducing capacity than luteolin under tested conditions.
Conclusions:
- The direct reduction of MTT by flavonoids is a significant confounding factor in MTT assays.
- Experimental conditions, including flavonoid concentration, incubation time, and solvent, influence this reaction.
- Results from MTT assays investigating flavonoid effects on cell growth may be artifactually altered by this chemical interaction.
