Unit 8: Cell viability by MTT assay - Subjective Questions

The MTT assay is a colorimetric method used to measure cell viability, proliferation, and cytotoxicity based on metabolic activity of living cells.

Full form: MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Basic principle:

• MTT is a yellow, water-soluble tetrazolium salt.
• Metabolically active (viable) cells contain mitochondrial dehydrogenase enzymes (primarily succinate dehydrogenase and NAD(P)H-dependent oxidoreductases).
• These enzymes reduce MTT by cleaving its tetrazolium ring to form insoluble purple formazan crystals.
• The amount of formazan produced is directly proportional to the number of viable, metabolically active cells.
• Dead cells lose enzymatic activity and cannot reduce MTT, so they produce no color.

The formazan is then solubilized and the absorbance measured spectrophotometrically to quantify viability.

Chemical reaction:

The yellow MTT tetrazolium salt is reduced by mitochondrial enzymes (dehydrogenases) in the presence of NAD(P)H to form purple formazan:

Key points:

• The reaction requires NADH/NADPH as reducing equivalents supplied by metabolically active cells.
• The tetrazolium ring of MTT is cleaved during reduction.

Color change:

• Before reaction: yellow solution (MTT).
• After reaction: purple/violet insoluble formazan crystals form inside and around living cells.
• The intensity of purple color increases with the number of viable cells.

The insoluble formazan is later dissolved using a solubilizing agent (DMSO or acidified isopropanol) to produce a homogeneous purple solution for absorbance measurement.

Step-by-step MTT assay procedure:

1. Cell seeding: Seed cells (typically to cells/well) into a 96-well microtiter plate and incubate at with until adherent.

2. Treatment: Add the test compound/drug at various concentrations. Include control wells (untreated) and blank wells (medium only). Incubate for the required time (24–72 h).

3. MTT addition: Add MTT solution (usually 5 mg/mL in PBS) to each well to a final concentration of ~0.5 mg/mL.

4. Incubation: Incubate for 3–4 hours at to allow formazan crystal formation.

5. Remove medium: Carefully aspirate the medium/MTT without disturbing crystals.

6. Solubilization: Add DMSO or acidified isopropanol to dissolve the purple formazan crystals.

7. Measurement: Read absorbance at 570 nm (reference ~630 nm) using a microplate reader (ELISA reader).

8. Analysis: Calculate percentage viability relative to control.

Percentage cell viability formula:

Since the absorbance is directly proportional to the number of viable cells, viability is calculated by comparing treated cells to control cells:

Explanation of terms:

• = absorbance of treated (test) wells containing cells + drug.
• = absorbance of untreated control wells (100% viable cells).
• = absorbance of blank wells (medium + MTT, no cells) to correct background.

Percentage cytotoxicity is derived as:

Note: The control absorbance represents maximum metabolic activity (taken as 100%). A lower sample absorbance indicates reduced viability, i.e., higher cytotoxicity of the test compound.

(Half Maximal Inhibitory Concentration):

It is defined as the concentration of a drug or compound required to inhibit 50% of cell growth/viability compared to untreated control.

Significance:

• It is a quantitative measure of a compound's potency in inhibiting cell viability.
• A lower indicates a more potent (more cytotoxic) compound.
• Used extensively in drug screening, anticancer research, and toxicology.

Determination from MTT data:

1. Perform MTT assay with a range of drug concentrations.
2. Calculate % viability for each concentration.
3. Plot a dose-response curve — % viability (Y-axis) vs. log concentration (X-axis).
4. The concentration corresponding to 50% viability on the curve is the .
5. Software tools (GraphPad Prism, Origin) perform non-linear regression to accurately calculate .

The curve is typically sigmoidal, allowing precise interpolation of the 50% inhibition point.

Comparison of viable and non-viable cells in MTT assay:

Key point:

• The assay does not directly count cells but measures metabolic activity as a proxy for viability.
• Only living, metabolically active cells contribute to the purple color, making the MTT assay an indirect measure of cell viability based on enzyme function.

Role of solubilizing agents (DMSO / acidified isopropanol):

After the MTT reduction reaction, purple formazan crystals are insoluble in aqueous medium and remain within the cells. To quantify them spectrophotometrically, they must be dissolved.

Functions:

• Dissolve formazan crystals: DMSO (dimethyl sulfoxide) or acidified isopropanol solubilizes the insoluble purple crystals into a homogeneous colored solution.
• Enable absorbance measurement: A uniform solution ensures accurate and reproducible optical density readings at 570 nm.
• Cell lysis: These solvents also lyse the cells, releasing intracellular formazan.

Choice of solvent:

• DMSO — most commonly used, rapid solubilization.
• Acidified isopropanol (0.04 N HCl in isopropanol) — reduces interference from phenol red in the medium.
• SDS-HCl — another alternative for solubilization.

Without solubilization, the crystals scatter light and give inaccurate readings.

Applications of the MTT assay:

• Cytotoxicity testing: Evaluating the toxic effects of drugs, chemicals, and nanoparticles on cells.
• Anticancer drug screening: Determining the efficacy and of potential anticancer agents on cancer cell lines.
• Cell proliferation studies: Measuring the growth response of cells to growth factors or media conditions.
• Drug sensitivity/resistance testing: Assessing how cancer cells respond to chemotherapeutic agents.
• Biocompatibility testing: Evaluating the safety of biomaterials and implants on cultured cells.
• Toxicology studies: Screening environmental toxins and pollutants.
• Radiation biology: Assessing effects of radiation on cell survival.
• Herbal/natural product research: Testing bioactivity of plant extracts.

Advantage: It is rapid, high-throughput (using 96-well plates), quantitative, and cost-effective, making it a standard tool in pharmacology, oncology, and toxicology.

Advantages of MTT assay:

• Simple and rapid to perform.
• High-throughput — many samples in 96-well format.
• Quantitative — measurable absorbance directly correlates with viability.
• Cost-effective and requires no radioactive materials.
• Sensitive to metabolic changes in cells.

Limitations of MTT assay:

• Endpoint assay: Cells must be destroyed (lysed) to solubilize formazan — not suitable for continuous monitoring.
• Formazan is insoluble: Requires a solubilization step, adding a step and possible error.
• Metabolism-dependent: Measures metabolic activity, not actual cell number — results can vary if metabolism changes without cell death.
• Interference: Test compounds, pH, glucose levels, and phenol red can affect readings.
• Cytotoxicity of DMSO: Excess solvent can affect results.
• Not suitable for non-adherent cells without modification.

Due to these limitations, alternatives like MTS, XTT, and WST assays (which produce soluble formazan) are sometimes preferred.

Comparison of MTT and Trypan Blue assays:

Summary:

• MTT is preferred for large-scale, quantitative cytotoxicity screening.
• Trypan Blue is quick for direct cell counting but is subjective and labor-intensive.

Absorbance at 570 nm:

• The purple formazan product has its maximum absorbance (absorption peak, ) at approximately 570 nm.
• Measuring at this wavelength gives the highest sensitivity and directly correlates with formazan concentration (and hence viable cell number).
• According to the Beer-Lambert law:
  
  where = absorbance, = molar absorptivity, = formazan concentration, = path length. Since is maximal near 570 nm, absorbance is most representative of formazan amount.

Role of reference wavelength (~630–690 nm):

• The reference wavelength is where formazan does not absorb.
• It corrects for background interference from cell debris, plate imperfections, and non-specific turbidity.
• Corrected absorbance = .
• This improves accuracy and reproducibility of the measurement.

Thus, dual-wavelength measurement ensures the signal truly reflects the formazan produced by viable cells.

Importance of controls and blanks:

Controls and blanks are essential for accurate, reliable, and reproducible MTT results, allowing correction of background signals and meaningful comparison.

Types of controls:

• Negative control (untreated cells): Cells with medium only (no drug). Represents 100% viability and maximum absorbance — the baseline for comparison.

• Positive control: Cells treated with a known cytotoxic agent (e.g., a standard drug). Confirms the assay detects reduced viability correctly.

• Blank (medium control): Wells containing medium + MTT but no cells. Corrects for background absorbance of the medium, MTT, and solvent.

• Vehicle/solvent control: Cells treated with the solvent used to dissolve the drug (e.g., DMSO) at the same concentration, to rule out solvent toxicity.

Purpose:

• Blank subtraction removes non-cellular background.
• Controls normalize data and validate assay performance.
• Solvent controls ensure toxicity is attributed to the test compound, not the vehicle.

Significance of mitochondrial dehydrogenase enzymes:

The MTT assay fundamentally relies on the activity of these enzymes present in living, metabolically active cells.

Key points:

• Location: These oxidoreductase enzymes are located mainly in the mitochondria (and partly in the cytoplasm and endoplasmic reticulum).
• Function in assay: They catalyze the reduction of the tetrazolium salt (MTT) into formazan using electrons from NADH and NADPH.
• Indicator of viability: Only viable cells with functional mitochondria possess active dehydrogenases; therefore, enzyme activity is a direct indicator of cell viability and metabolic health.
• Proportionality: The amount of formazan formed is proportional to the level of enzymatic activity, which in turn reflects the number of living cells.

Reaction summary:

Conclusion: Loss of enzyme activity (in dead/dying cells) means no formazan formation, making these enzymes the biochemical basis of the assay.

Given data:

Step 1: Apply the cell viability formula:

Step 2: Substitute values:

Step 3: Calculate % cytotoxicity:

Result:

• Cell viability = 36%
• Cytotoxicity = 64%

Interpretation: The test compound reduced viability substantially (only 36% cells remain viable), indicating it is significantly cytotoxic at the tested concentration.

Dose-response curve:

A dose-response curve is a graphical representation showing the relationship between the concentration (dose) of a test compound and the biological response (here, % cell viability).

Construction:

1. Treat cells with a series of increasing concentrations of the compound.
2. Perform MTT assay and calculate % viability at each concentration.
3. Plot the graph:
   • X-axis: Concentration (often log scale of concentration).
   • Y-axis: % Cell viability.
4. The resulting curve is typically sigmoidal (S-shaped).

Interpretation:

• High viability at low doses: minimal toxicity.
• Decreasing viability with increasing dose: dose-dependent cytotoxicity.
• The midpoint (50% viability) gives the value.
• Steepness of the curve reflects the sensitivity of cells to the drug.

Significance:

• Determines potency () and efficacy of a compound.
• Helps establish safe vs. toxic concentration ranges for drugs.

Factors affecting MTT assay accuracy:

• Cell density (seeding number): Too few or too many cells give unreliable results; optimal density is essential for linear correlation between viability and absorbance.

• MTT concentration and incubation time: Insufficient MTT or short incubation yields low formazan; excess may cause toxicity. Standard: ~0.5 mg/mL, 3–4 h.

• Metabolic state of cells: Changes in metabolism (glucose, pH) can alter formazan production independent of cell number.

• Presence of phenol red / medium components: Can interfere with absorbance readings.

• Test compound interference: Compounds that are colored or that reduce MTT chemically (e.g., antioxidants, reducing agents) cause false readings.

• Complete solubilization of formazan: Incomplete dissolution causes errors; thorough mixing with DMSO is needed.

• Wavelength selection: Using correct 570 nm and reference wavelength.

• Temperature and CO₂: Improper incubation conditions affect enzyme activity.

• Edge effects / evaporation in 96-well plates: Outer wells may evaporate faster, skewing results.

Conclusion: Standardization and proper controls minimize these errors.

Comparison of MTT vs. XTT/MTS assays:

Key differences:

• MTT produces insoluble intracellular purple formazan requiring a solubilization step.
• XTT and MTS produce water-soluble formazan directly in the medium, eliminating the solubilization step, making them more convenient for high-throughput screening.

Despite being older, MTT remains widely used due to low cost and reliability.

MTT assay in anticancer drug screening:

The MTT assay is a primary in-vitro screening tool to evaluate the cytotoxic potential of candidate anticancer compounds against cancer cell lines.

Example workflow:

1. Cell line selection: Choose relevant cancer cell lines (e.g., MCF-7 for breast cancer, HeLa for cervical cancer).

2. Seeding: Plate cells in a 96-well plate at optimized density.

3. Drug treatment: Add the candidate drug at multiple concentrations (e.g., 1, 5, 10, 25, 50, 100 µM), with untreated controls.

4. Incubation: Incubate 24–72 h.

5. MTT addition & incubation: Add MTT, incubate 3–4 h for formazan formation.

6. Solubilization & reading: Dissolve crystals in DMSO; read at 570 nm.

7. Data analysis: Calculate % viability, plot dose-response curve, determine .

Interpretation:

• A low indicates a potent anticancer candidate.
• Selectivity is checked by comparing effects on cancer vs. normal cells.

Significance: Enables rapid, quantitative ranking of drug candidates before advancing to animal studies.

Precautions during MTT assay:

• Sterility: Maintain aseptic conditions throughout to prevent contamination.

• MTT handling: MTT is light-sensitive and toxic — prepare fresh, store in the dark, and handle with gloves.

• Uniform seeding: Ensure even, consistent cell density across all wells for reproducibility.

• Avoid edge wells / evaporation: Use outer wells with PBS or avoid using them to prevent edge effects and evaporation.

• Gentle aspiration: Remove medium carefully without disturbing/aspirating formazan crystals or dislodging adherent cells.

• Complete solubilization: Mix thoroughly with DMSO to fully dissolve crystals; avoid air bubbles that interfere with reading.

• Optimal incubation: Follow correct MTT incubation time (3–4 h); over/under-incubation affects formazan yield.

• Proper controls & blanks: Always include blank, negative, and solvent controls.

• Avoid interfering compounds: Be cautious with colored or reducing test compounds that skew absorbance.

• Consistent timing: Read absorbance promptly and consistently across plates.

Result: Following these precautions ensures accurate, reproducible viability measurements.

Theoretical basis:

The MTT assay assumes that metabolic activity is directly proportional to the number of viable cells. Living cells with functional mitochondria continuously reduce MTT to formazan via NAD(P)H-dependent dehydrogenases. Since dead cells lack this activity, the total formazan produced serves as a surrogate measure of viable cell number.

Situations giving misleading results:

• Altered metabolism without cell death: Drugs may increase or decrease metabolic rate independent of viability, over/underestimating cell number.

• Quiescent/dormant cells: Metabolically inactive but living cells give low signal, falsely indicating low viability.

• Hyper-metabolic stress response: Stressed cells may temporarily increase MTT reduction, overestimating viability.

• Chemical interference: Reducing agents/antioxidants directly reduce MTT, giving false high readings.

• Glucose concentration effects: High glucose can enhance formazan production irrespective of cell number.

• Non-mitochondrial reduction: Some MTT reduction occurs outside mitochondria, complicating interpretation.

Conclusion: Because MTT measures metabolic activity, not direct cell count, results should be confirmed with complementary assays (e.g., Trypan blue, LDH release) when interpreting cytotoxicity.