Unit 8: Cell viability by MTT assay

I. Orientation: Cell Viability and the Principle of Colorimetric Assays

Cell viability assays quantify the proportion of living, metabolically active cells in a population, a core measurement in cytotoxicity testing, drug screening, and proliferation studies. The MTT assay (developed by Tim Mosmann, 1983) is the archetypal colorimetric method: it converts a metabolic signal into an optical density readable on a plate reader.

• Viability defined: the fraction of cells retaining intact membranes and functioning metabolism; MTT reports metabolic activity, not membrane integrity directly.
• Metabolic readout: viable cells reduce a soluble tetrazolium salt to an insoluble coloured product; dead cells cannot.
• Endpoint, not real-time: MTT is destructive — cells are lysed to release the product, so each well yields a single time-point value.
• Quantitative basis: signal (absorbance) is assumed proportional to the number of viable cells within a working range, enabling relative comparison against untreated controls.
• Format: performed in 96-well microplates, allowing many concentrations and replicates in parallel.
• Key reagent: MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow water-soluble salt.

II. The MTT Assay: Principle, Protocol and Interpretation

A. Purpose and Biochemical Principle

The assay measures viable cell number by exploiting the reduction of MTT to a coloured formazan by living cells.

• Reduction reaction: the yellow tetrazolium ring of MTT is cleaved to form purple, water-insoluble formazan crystals.
• Enzymatic driver: reduction is catalysed largely by mitochondrial and cytosolic dehydrogenases (e.g., succinate dehydrogenase) using NAD(P)H as the electron donor.
  • NAD(P)H dependence: signal reflects the metabolic/redox state of the cell, not merely its presence.
  • Cofactor link: only cells with active electron-transport and reducing power generate colour.
• Colour change: yellow → purple, with formazan absorbing strongly around 570 nm.
• Proportionality: more viable, metabolically active cells → more formazan → higher absorbance.

MTT (yellow, soluble)  --[dehydrogenase / NAD(P)H]-->  Formazan (purple, insoluble)

• Symbols/terms:
  • MTT = tetrazolium salt substrate.
  • NAD(P)H = reduced nicotinamide adenine dinucleotide (phosphate), electron donor.
  • Formazan = coloured product; quantity ∝ reducing activity.

B. Reagents, Equipment and Cell Preparation

The assay requires standardised reagents and healthy, accurately counted cells to yield reliable data.

• MTT stock: typically 5 mg/mL in phosphate-buffered saline (PBS), filter-sterilised, stored protected from light at 4 °C.
• Solubilising agent: DMSO or acidified isopropanol (or SDS-HCl) to dissolve formazan crystals into a homogeneous coloured solution before reading.
• Culture medium: phenol-red-free medium preferred, as phenol red absorbs near the read wavelength and inflates background.
• Equipment: CO₂ incubator (37 °C, 5 % CO₂), laminar flow hood, microplate spectrophotometer (ELISA reader), haemocytometer or automated counter.
• Cell preparation:
  • Counting: determine viable cells (trypan blue exclusion) and seed a defined number per well.
  • Seeding density: commonly 5,000–10,000 cells/well in 100 µL, chosen so cells remain sub-confluent and within the linear range at endpoint.
  • Attachment: adherent cells incubated 24 h to attach before treatment.

C. Evaluation of Cell Viability by MTT Assay

This is the operational core: exposing cells to a test agent, developing colour, and converting absorbance into a viability percentage.

• Step 1 — Seed: dispense equal cell numbers into 96 wells; include blank (medium only), untreated control, and treated wells.
• Step 2 — Treat: add test compound over a concentration series (e.g., serial dilutions), plus vehicle control (e.g., matched DMSO %). Incubate a set period (commonly 24–72 h).
• Step 3 — Add MTT: add MTT to each well (final ≈ 0.5 mg/mL); incubate 3–4 h at 37 °C to allow formazan formation.
• Step 4 — Remove and solubilise: aspirate medium carefully (crystals are insoluble and adherent), add solubiliser (e.g., 100 µL DMSO), and mix until crystals dissolve.
• Step 5 — Read: measure absorbance at 570 nm with a reference at 630–690 nm to subtract non-specific scatter.
• Step 6 — Calculate: express treated signal relative to control after blank subtraction.

% Cell viability = [ (A_sample − A_blank) / (A_control − A_blank) ] × 100

• Symbols:
  • A_sample = absorbance of treated well.
  • A_control = absorbance of untreated (vehicle) well.
  • A_blank = absorbance of medium-only well (background).

Worked example:

• A_control = 0.90, A_blank = 0.05, A_sample (drug at 10 µM) = 0.48.
• Viability = (0.48 − 0.05) / (0.90 − 0.05) × 100 = 0.43 / 0.85 × 100 ≈ 50.6 %.
• Interpretation: at 10 µM the agent halves viability, so this concentration approximates the IC₅₀ (concentration causing 50 % reduction).

D. Controls, Replicates and Data Analysis

Meaningful viability values depend on properly designed controls and statistical treatment of replicates.

• Blank well: medium + MTT + solubiliser, no cells — corrects for reagent/medium absorbance.
• Negative (untreated) control: cells with vehicle only — defines 100 % viability baseline.
• Vehicle control: matches solvent concentration (e.g., 0.1 % DMSO) so solvent toxicity is not misread as drug effect.
• Positive control: a known cytotoxic agent confirms the assay detects cell death.
• Replicates: ≥ 3 technical wells per condition and independent biological repeats; report mean ± SD or SEM.
• Dose–response: plot % viability against log concentration; fit a sigmoidal curve to derive IC₅₀.

E. Applications

MTT is a versatile screening tool wherever a metabolic proxy for live-cell number suffices.

• Cytotoxicity screening: ranking candidate drugs, nanoparticles, or environmental toxicants by IC₅₀.
• Anticancer research: measuring tumour-cell killing across concentrations and cell lines.
• Proliferation studies: tracking increase in viable cells over time under growth factors or media.
• Biocompatibility testing: assessing biomaterials and scaffolds for tissue engineering.
• High-throughput format: 96- (or 384-) well plates enable large compound libraries in one run.

F. Advantages and Limitations

The assay's popularity reflects real strengths, but its indirect readout imposes clear caveats.

1. Advantages:
   • Simplicity and cost: few reagents, standard plate reader, no radioactivity.
   • Throughput: parallel testing of many wells; quantitative and reproducible.
   • Sensitivity: detects modest changes in viable-cell number within the linear range.
2. Limitations:
   • Metabolic, not count-based: agents that alter metabolism without killing cells skew results (false high or low).
   • Endpoint/destructive: cells lysed during solubilisation, so no kinetic monitoring of the same well.
   • Interference: reducing compounds (e.g., ascorbate), phenol red, and some drugs directly reduce MTT, causing artefacts.
   • Solubility step: insoluble formazan needs a solubilisation stage, adding a variable and requiring aspiration that can dislodge cells.
   • Density dependence: signal only linear across a limited cell-number window; over-confluent wells plateau.

III. Related Tetrazolium and Comparative Considerations

A. Formazan Chemistry and Reading Conditions

Accurate absorbance depends on complete, uniform solubilisation and correct optics.

• Wavelength choice: read at the formazan peak (≈ 570 nm); the exact peak shifts slightly with solvent.
• Reference wavelength: subtract absorbance at 630–690 nm to remove debris/scatter and well imperfections.
• Mixing: ensure crystals fully dissolve; undissolved particles cause erratic optical density.
• Timing: read promptly after solubilisation, as signal can drift with prolonged standing or light exposure.

B. MTT versus Newer Tetrazolium Salts (MTS/XTT/WST)

Later tetrazolium reagents were designed to remove the solubilisation step that limits MTT.

1. MTT:
   • Product: insoluble intracellular formazan — requires a separate solubilisation/aspiration step.
   • Site: reduced largely inside cells.
2. MTS / XTT / WST-1:
   • Product: water-soluble formazan — no solubilisation step, add-and-read.
   • Electron coupling: use an intermediate electron acceptor (e.g., PMS) and are often reduced at the cell surface.
   • Trade-off: more convenient and less disruptive, but reagents are costlier and can be less stable than MTT.

C. MTT versus Direct Viability Assays

MTT reports metabolism; other methods report membrane integrity or enzyme leakage, and the distinction matters for interpretation.

1. MTT (metabolic activity):
   • Reports: reducing/enzymatic capacity of live cells.
   • Blind to: early membrane damage if metabolism persists.
2. Dye-exclusion / release assays (e.g., trypan blue, LDH):
   • Trypan blue: stains cells with compromised membranes — a direct dead/live count.
   • LDH release: measures cytoplasmic enzyme leaking from lysed cells — a cytotoxicity (membrane-damage) endpoint.
   • Complementarity: pairing MTT with an LDH or dye-exclusion assay distinguishes metabolic inhibition from true cell death.