The primary disadvantage of the MTT assay stems from the water-insoluble nature of its formazan product, which necessitates an additional solubilization step using organic solvents like dimethyl sulfoxide (DMSO) or isopropanol before absorbance measurement.
Key Disadvantages of the MTT Assay
While widely used for assessing cell viability and proliferation, the MTT assay presents several limitations that researchers must consider. These drawbacks can impact the accuracy, reliability, and comparability of results.
Here's a breakdown of the main disadvantages:
-
Solubility and Processing Requirements:
- A significant drawback is that the MTT formazan, the purple reaction product, is insoluble in water and forms needle-shaped crystals within the cells.
- This insolubility necessitates an extra step of adding an organic solvent, such as dimethyl sulfoxide (DMSO) or isopropanol, to solubilize these crystals prior to measuring absorbance. This additional step can introduce variability and increase handling time.
-
Metabolic Activity vs. Cell Number:
- The MTT assay measures the activity of mitochondrial dehydrogenases, which are enzymes indicative of cellular metabolic activity. It does not directly quantify cell number or cell membrane integrity.
- Consequently, changes in cellular metabolic state (e.g., cells entering quiescence or undergoing stress) can affect the MTT signal even if the number of viable cells remains constant, potentially leading to misinterpretations.
-
Interference from Assay Components and Test Compounds:
- Various substances commonly found in cell culture, such as phenol red in culture media, or colored test compounds, can absorb light at similar wavelengths to formazan, leading to false readings.
- The pH of the medium can also influence the MTT reduction, potentially causing non-enzymatic reduction of MTT and inaccurate results.
-
Cell Type Variability:
- Different cell lines and primary cells can exhibit varying levels of mitochondrial dehydrogenase activity, even when viable and at similar cell densities. This inherent variability makes it challenging to compare results directly across diverse cell types without proper internal controls and calibration.
-
Toxicity of Solubilizing Agents:
- While essential for solubilization, the organic solvents like DMSO can be cytotoxic themselves, especially at higher concentrations or prolonged exposure. This can be a concern if downstream analyses are planned on the cells or if the solvent residue impacts the experimental environment.
-
Non-Linearity at High Cell Densities:
- At very high cell concentrations, the conversion of MTT to formazan may not be linear with cell number due to substrate depletion or saturation of enzyme activity. This can lead to an underestimation of viability in dense cultures.
-
Multiple Step Procedure and Error Potential:
- The assay involves several steps, including incubation, media removal, addition of MTT reagent, further incubation, removal of supernatant, and finally the addition of solubilization solution. Each step is a potential source of experimental error, such as incomplete washing, uneven reagent distribution, or incomplete formazan dissolution.
Summary of Disadvantages
The following table provides a concise overview of the key disadvantages associated with the MTT assay:
| Disadvantage Category | Specific Issue |
|---|---|
| Product Solubility | MTT formazan is water-insoluble, requiring an additional step to solubilize purple, needle-shaped crystals using organic solvents (e.g., DMSO, isopropanol). |
| Assay Readout Mechanism | Measures metabolic activity (mitochondrial dehydrogenase) rather than direct cell count or proliferation, leading to potential misinterpretations. |
| Interference | Colored compounds, media components (e.g., phenol red), and pH changes can interfere with absorbance readings, causing false positives or negatives. |
| Cell Type Dependency | Metabolic activity varies significantly between different cell types, impacting assay sensitivity and comparability across diverse samples. |
| Solvent Toxicity | Organic solvents used for formazan solubilization can be cytotoxic, posing risks for downstream experiments or sensitive cells. |
| Linearity Limitations | The assay may exhibit non-linear signal responses at very high cell densities, leading to inaccurate viability assessments. |
| Methodological Complexity | Multiple handling steps increase the potential for experimental variability and error during the assay procedure. |
