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Mastering Cell Viability: The Chemicals That Interfere with Tetrazolium Assays (MTT/MTS)
- CLYTE research team
- 5 hours ago
- 5 min read
In the realm of cytotoxicity and cell proliferation research, tetrazolium-based assays—most notably the MTT assay—stand as the gold standard for high-throughput screening. They are cost-effective, rapid, and generally reliable. However, "generally" is the operative word. For researchers investigating novel therapeutics, natural products, or specific chemical classes, the MTT assay can become a minefield of artifacts and false data.
This article delves into the science of why certain chemicals interfere with tetrazolium assays, analyzing the mechanisms behind false positives and false negatives, and providing actionable strategies to ensure your data remains robust.
The Chemistry of Deception: How MTT Works (and Fails)
To understand interference, we must first understand the assay's core mechanism. The MTT assay relies on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into an insoluble, purple formazan product. Theoretically, this reduction is catalyzed exclusively by mitochondrial succinate dehydrogenases in metabolically active cells. Thus, the intensity of the purple color (absorbance) should correlate directly with the number of viable cells.
The problem arises when non-cellular chemical agents hijack this reduction process or destroy the resulting signal, leading to data that reflects chemical reactivity rather than cell viability.
Type 1: The False Positives (Direct Reduction)
The most common form of interference is the "False Positive," where the test compound reduces the tetrazolium salt directly, creating a purple signal even in the absence of live cells. This makes toxic compounds appear non-toxic or even proliferative.
1. Thiol-Containing Compounds
Chemicals possessing free thiol (sulfhydryl) groups are notorious for reducing tetrazolium salts. Research indicates that compounds such as beta-mercaptoethanol, dithiothreitol (DTT), and N-acetyl-L-cysteine (NAC) can reduce MTT in a dose-dependent manner without any cellular enzymes present. If your drug screening involves thiol-donors or glutathione precursors, your viability data may be artificially inflated.
2. Vitamins and Antioxidants
Antioxidants are celebrated for their cytoprotective properties, but they are enemies of the MTT assay.
Ascorbic Acid (Vitamin C): A potent reducing agent that rapidly converts MTT to formazan.
Vitamin E (α-Tocopherol): Specifically, the succinate form has been shown to interfere, though often less aggressively than Vitamin C.
Kaempferol and Quercetin: These abundant flavonoids (found in plant extracts) are powerful reducers. Studies have shown that the number of phenolic hydroxyl groups on the flavonoid ring correlates with the intensity of the non-enzymatic reduction.
3. Plant Extracts
The screening of "natural products" is a frequent source of error. Extracts rich in polyphenols (like green tea catechins or epigallocatechin gallate - EGCG) can interact with MTT. When testing plant extracts, high background absorbance in cell-free control wells is a tell-tale sign of interference.
Type 2: The False Negatives (Signal Degradation)
While less discussed, some chemicals can suppress the signal, leading to "False Negatives" where viable cells appear dead.
Porphyrins and Photosensitizers
Recent findings published in the International Journal of Molecular Sciences highlight a distinct mechanism of interference by porphyrin-related compounds.
Zinc Protoporphyrin (ZnPP) and Protoporphyrin IX (PPIX) were found to significantly reduce the color response of MTT formazan.
Mechanism: Unlike antioxidants that create formazan, these compounds appear to promote the rapid degradation of the formazan dye through photosensitizing properties when exposed to light. This results in an underestimation of cell viability.
Type 3: Technical and Environmental Artifacts
Interference isn't always chemical; it can be environmental.
pH Changes: The reduction of tetrazolium salts is pH-dependent. Acidic environments can suppress formazan formation, while alkaline conditions can enhance spontaneous reduction.
Serum and Media: High concentrations of serum (e.g., Fetal Bovine Serum) or specific media formulations (like those containing phenol red) can interact with flavonoids to amplify their reducing power, exacerbating false positives.
Solubility Issues: Incomplete solubilization of formazan crystals by solvents (DMSO, SDS/HCl) can lead to high variability and "spikes" in absorbance data.
Best Practices: How to Detect and Avoid Interference
The Golden Rule: Cell-Free Controls: Always include a set of wells containing the test compound and MTT reagent without cells. If these wells turn purple (for MTT) or show absorbance changes, your compound is interfering.
Wash the Cells: If your compound interferes, try washing the cells with PBS to remove the medium containing the drug before adding the MTT reagent. Note: This may not work if the compound has accumulated intracellularly.
Use Alternative Assays: If interference is detected, switch to an assay with a different mechanism:
ATP Assays (Luminescence): Measures cellular ATP content; generally less susceptible to chemical reduction artifacts.
LDH Release (Enzymatic): Measures membrane integrity via lactate dehydrogenase leakage.
Sulforhodamine B (SRB): A protein-binding assay that is not dependent on redox activity.
Protect from Light: To avoid photosensitive degradation (as seen with porphyrins), perform incubations in the dark.
Conclusion
The MTT assay remains a vital tool in the scientific arsenal, but it is not infallible. By recognizing the chemical signatures of interference—specifically thiols, antioxidants, and photosensitizers—researchers can avoid the costly trap of publishing artifacts as results. Always validate "too good to be true" viability data with alternative methods.
MTT / Cell Viability Frequently Asked Questions (FAQ)
What are common MTT assay problems?
The most frequent issues researchers encounter include high background absorbance, which often stems from protein precipitation or phenol red interference. Inconsistent data between replicates is another common problem, usually caused by pipetting errors, uneven cell seeding (edge effects), or incomplete solubilization of the formazan crystals. Additionally, false positives frequently occur when testing compounds with reducing properties (like antioxidants) that convert MTT to formazan non-enzymatically.
What are the limitations of the MTT assay?
While popular, the MTT assay has significant limitations:
Metabolic Dependence: It measures metabolic activity, not specifically cell death. Treatments that affect mitochondrial function without killing the cell (metabolic inhibitors) can skew results.
Chemical Interference: As detailed in this article, reducing agents, thiols, and colored compounds can interact with the reagent, leading to false data.
End-Point Only: It is a destructive assay; the cells are killed during the solubilization step, preventing further analysis of the same sample.
Solubility Steps: The requirement to dissolve formazan crystals introduces an extra handling step that can increase experimental error compared to soluble reagents like MTS or WST-1.
Why do we use DMSO in MTT assay?
DMSO (Dimethyl Sulfoxide) is used as a solvent to dissolve the purple formazan crystals. When live cells reduce the yellow MTT tetrazolium salt, the resulting product is insoluble formazan, which precipitates inside and on the surface of the cells. To measure the color intensity using a spectrophotometer (plate reader), these solid crystals must be fully dissolved into a liquid solution. DMSO is highly effective at solubilizing these crystals rapidly.
What reduces MTT?
In a biological context, MTT is primarily reduced by mitochondrial dehydrogenase enzymes (using NADH and NADPH as cofactors) found in metabolically active cells. However, in a chemical context, MTT can be reduced by any strong reducing agent. Common non-biological reducers include Vitamin C (ascorbic acid), Vitamin E, sulfhydryl-containing compounds (like Glutathione or DTT), and various flavonoids found in plant extracts. These chemical reductions are the primary cause of assay interference.
