MTT Assay Protocol: Guide to Measuring Cell Viability & Proliferation

CLYTE research team
13 minutes ago
4 min read

The MTT assay is one of the most widely used methods in cell biology for measuring cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. Whether you are testing the toxicity of a new drug or evaluating the growth rate of primary cells, mastering this protocol is essential for generating reproducible data.

Ask Sophie about how you can do MTT assay with your specific cells and reagents!

You might also be interested in this Migration vs Proliferation article!

This guide provides a detailed, optimized protocol for the MTT assay, specifically tailored for Primary Human Dermal Fibroblasts (HDFs) based on laboratory standards. It also integrates industry best practices to ensure your results are accurate and publication-ready.

1. Principle of the MTT Assay

The MTT assay is a colorimetric assay for assessing cell metabolic activity. It relies on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow tetrazolium salt, into purple formazan crystals.

Mechanism: NAD(P)H-dependent cellular oxidoreductase enzymes (primarily in the mitochondria of living cells) reduce the yellow MTT dye into insoluble purple formazan.
Significance: Since this conversion only occurs in metabolically active cells, the amount of formazan produced is directly proportional to the number of viable cells .
Quantification: The crystals are solubilized (usually with DMSO), and the resulting colored solution is quantified by measuring absorbance using a spectrophotometer.

2. Materials & Reagents

To perform a high-quality MTT assay, ensure you have the following materials prepared. This specific list is optimized for adherent cells like HDFs in a 48-well plate format.

Biological Materials

Primary Human Dermal Fibroblasts (HDFs): (Cat# PCS-201-012).
Growth Medium: PCS-030 (supplemented with 2% serum for seeding).

Reagents

MTT Reagent: 5 mg/mL in Phosphate-Buffered Saline (PBS). Must be filter-sterilized and protected from light.
Solubilization Agent: Dimethyl sulfoxide (DMSO).
Test Product: Prepared in serum-free media (SFM) at various concentrations.
Phosphate-Buffered Saline (PBS): For washing cells.

Equipment

48-well cell culture plates.
Microscope: To observe morphology.
Spectrophotometer/Plate Reader: Capable of reading absorbance at 540 nm.

3. Step-by-Step Protocol

This protocol evaluates cell proliferation over multiple time points (24h, 48h, 120h) to assess product influence on growth.

Phase 1: Cell Seeding (Day 0)

The goal is to seed cells at a density that allows for exponential growth without reaching overconfluency before the assay endpoint.

Prepare Cell Suspension: Trypsinize HDFs and prepare a suspension in low-serum medium (2%).
Seeding Density: Seed 10,000 cells/well into a 48-well plate.
- Volume: Add 500 µL of cell suspension per well.
Incubation: Incubate plates at 37°C in 5% CO₂ for 24 hours to allow cell adhesion.

Note: Maintaining a consistent seeding density is critical. Variations here will directly skew your absorbance readings.

Phase 2: Treatment (Day 1)

Once cells have adhered, treat them with your test product.

Prepare Concentrations: Dilute the product in serum-free media (with phenol red) to create 6 concentrations: 10, 5, 1, 0.5, 0.1, and 0.05 mg/mL.
Apply Treatment:
- Carefully remove the old media from each well.
- Test Wells: Add 500 µL of the specific product concentration.
- Controls: Include Positive Control (2% serum media) and Negative Control (Serum-Free Media) wells, adding 500 µL of fresh medium to each.
- Replicates: Use n=4 wells for each group to ensure statistical significance.
Incubation: Return plates to the incubator (37°C, 5% CO₂).

Phase 3: MTT Assay Procedure (Days 2, 3, and 6)

Perform the assay at your designated time points (e.g., 24h, 48h, 120h post-treatment).

Observation: Check cells under a microscope for morphological changes or toxicity signs.
Wash: Remove treatment media and gently wash wells with PBS.
MTT Addition:
- Add 250 µL of MTT solution (diluted to 0.5 mg/mL in phenol red-free, serum-free medium) to each well.
- Tip: Light sensitivity! Keep the MTT solution covered or in low light.
Incubation: Incubate for 2-3 hours at 37°C. Look for the formation of intracellular purple crystals.
Solubilization:
- Carefully remove the MTT solution (do not disturb the crystals at the bottom).
- Add 250 µL of DMSO to each well to dissolve the formazan crystals.
- Gently shake the plate to ensure complete dissolution.
Measurement: Measure absorbance at 540 nm using a plate reader.

Long-term Culture Note: For the 120h (Day 6) plate, refresh the media on Day 3 with freshly prepared product and control solutions to maintain nutrient levels.

4. Data Analysis & Calculation

To determine the effect of your product on cell proliferation, calculate the percentage of cell viability for each concentration.

Cell Viability (%) = (Absorbance of Treated Sample / Absorbance of Control) x 100

Background Correction: If you have blank wells (media only, no cells), subtract their average absorbance from all other readings before calculating viability.
Graphing: Plot % Viability (Y-axis) vs. Concentration (X-axis) to visualize the dose-response relationship.

5. Troubleshooting Common Issues

Issue	Possible Cause	Solution
High Background	Phenol red interference or protein precipitation	Use phenol red-free media for the MTT step. Ensure complete washing.
Low Absorbance	Low cell density or short incubation	Optimize seeding density (prevent <10,000 cells/well) or extend MTT incubation to 4 hours.
Variation in Replicates	Pipetting error or uneven evaporation	Use a multichannel pipette; ensure the incubator is humidified.
Precipitation	Product reacting with MTT	Wash cells thoroughly with PBS before adding MTT reagent.