Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons

Abstract

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.

Preparation of mouse anti‑human rotavirus VP7 monoclonal antibody and its protective effect on rotavirus infection

“…The MTT colorimetric method is used to detect the activity and growth rate of cells. The OD value was measured in the current study using a spectrophotometer at 578 nm to determine the number of surviving cells following an MTT assay (34,35). The results demonstrated that the mouse anti-human RV VP7 monoclonal antibody exhibited good neutralization activity, with a cell protection rate of 43.3% and a neutralization titer of 1:446.…”

Antioxidant, Antimicrobial, and Other Biological Properties of Pompia Juice

“…In our study, CVS was used to evaluate CaCo2 and SW48 cancer cells number when exposed to a preventive treatment with increasing concentration of citrus juices ( Figure S7 ), as this non-enzymatic assay allows the estimation of the number of viable cells, adherent and colonies, since the amount of dye absorbed depends on the total DNA content [ 38 ]. Differently, the MTT assay may result in either underestimation or overestimation of the fraction of live cells with the result depending on the type of cells [ 39 ].…”

Properties of novel surfactin‐derived biosurfactants obtained through solid‐phase synthesis

“…The MTT assay evaluates cell viability based on the reduction of a tetrazolium salt to formazan crystals as a result of cell metabolic activity and showed that challenge of Vero cells with the LPs 6, 7, and 8 peptides increased the amount of formazan produced (Figure A). This can be interpreted as an increase in the metabolic flux due to augmented mitochondrial respiration rates and/or cell redox stress, as discussed elsewhere . Thus, as a more direct test for cell integrity and viability, alternatively we used the sulforhodamine B assay, which binds nonspecifically to proteins within cells.…”

“…This can be interpreted as an increase in the metabolic flux due to augmented mitochondrial respiration rates and/or cell redox stress, as discussed elsewhere. 53,54 Thus, as a more direct test for cell integrity and viability, alternatively we used the FIGURE 4 Hemolytic assay with the novel peptide biosurfactants. Hemolysis was monitored for each peptide at different concentrations (up to 128 μM) as described in Section 2.…”