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Cytotoxicity Test by MTT Assay- Procedure, Analysis and Results

Cytotoxicity Test by MTT Assay- MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test is used to measure the cellular metabolic activity in mammalian cells as an indicator of cell viability, proliferation and cytotoxicity. Yellow water soluble MTT is metabolically reduced in viable cells to a blue-violet insoluble formazan. The number of viable cells correlated to the color intensity determined photographic measurement after dissolving the formazan in alcohol.

Cytotoxicity Test by MTT Assay
Cytotoxicity Test by MTT


MTT is a popular cell proliferation assay that is used to assess the effects of various compounds on cell proliferation. The MTT assay is based on the principle that cells convert a yellow tetrazolium salt, MTT, into an insoluble purple formazan product. The amount of formazan produced is directly proportional to the number of living cells in the culture and can be quantified by measuring the absorbance at 550 nm.

The MTT assay has been used for many years and is a well-established method for measuring cell proliferation. However, there are some drawbacks to the assay including its reliance on a single endpoint (absorbance at 570 nm), its sensitivity to changes in pH and temperature, and its relatively long incubation time (3-4 hours).

Table of Content

Cytotoxicity Test by MTT Assay


  1. MTT test can be done using mammalian cell lines.
  2. Prepare the cell culture from the frozen stock or T-Flask.
  3. Prepare culture media with 10% FBS, 1% L-glutamine and 1% Antibiotic. Keep it in sterile conditions.
  4. Check the confluency and morphology of the flask using inverted microscope.
  5. Once 70% reached prepare for subculture.
  6. Wipe laminar air flow chamber with 70% ethanol and autoclave the pipette tips.
  7. Wipe chemical bottles and pack with 70% ethanol after thawing.

Preparation for Subculture for Cytotoxicity Assay

  1. Remove the old media from the flask and discard in beaker. Wash the T flak with PBS at least two times.
  2. Add 1 mL of Trypsin- EDTA and keep the flak at 37°C for 10 min.
  3. Gently shake the flask under the laminar air flow until the cell gets detached. Check the progress in the Inverted microscope.
  4. If cells are detached, aspirate the medium from the flask in the centrifuge tube. Add 5 mL fresh media and centrifuge at 1200 rpm for 10 min.
  5. After centrifugation clear pallet will be visible in the bottom of the tubes.
  6. Remove the supernatant carefully without disturbing the pallet.
  7. Add fresh medium up to 5 mL to remove the traces of Trypsin- EDTA.
  8. Again centrifuge it at 1000 rpm for 10 min.
  9. Discard the supernatant, and add required amount of fresh medium.
  10. Suspend the pallet.
  11. For subculture add 5-20mL of complete medium in the T flask (25-cm² flask or 75-cm² ) and add prepared culture of the cells from the centrifuge tube.
  12. 1 flask can be split in 3 to 5 flask.

Transferring cells in to 96 well plates

  1. After 70-80% confluency, remove the cells from culture flasks by enzymatic digestion (Trypsin-EDTA) and centrifuge the cell suspension at 1200 rpm for 10 minute.
  2. Re-suspend the cells in culture medium at a density of 1-3 x 105cells/mL.
  3. Prepare the plate map (96-well microtiter plate) for blank, negative control and for test item; dispense 100µL of culture medium into the specific wells and served as blank and seed 100µL of cells into 96-well plate as per plate map prepared for negative control and test item.
  4. Incubate the cells further for 24h at 5% CO2 at 37°C with >90% humidity.

Test Item Exposure

  1. After 24h of incubation, add 100µL of test item, positive control, negative control and blank in duplicates into the respective wells of the 96-well plate based on plate map.
  2. Add eight to ten different concentrations of the test item. Incubate the cells for 24h at 5% CO2 at 37°C with >90% humidity.


  1. Examine the cells after 24h treatment using phase contrast microscope (Fluorescence microscopy) to check the morphological changes.
  2. Remove the culture medium from the wells,  add 50µL of MTT solution (1mg/mL) to each well and the plate
  3. Incubate it further for 2h at 5% CO2 at 37°C with >90% humidity.
  4. Descend MTT solution and 100µL of isopropanol in each well.
  5. The color shift to yellow is acidic pH and the color shift to magenta to purple is alkaline pH.
  6. Absorbance will be measured at 550nm using Microplate reader to ensure the cell viability.

Analysis-Cytotoxicity Test by MTT Assay

The Optical Density (OD) at 550nm will be used to calculate the percentage of viability results using the following equation:

Viability % = 100 x OD550e/OD 550b

             Where, OD550e – Mean value of the measured optical density of the test item.

             OD550b – Mean value of the measured optical density of the negative control.


The results will be evaluated based on the criteria of

  1. A decrease in the number of living cells results in reduction in the metabolic activity in the test sample. This decrease directly correlates to the amount of blue-violet formazan as evident.
  2. No cytotoxicity shall be observed in negative controls.
  3. Test item at higher concentration may exhibit cytotoxicity (in vitro cytotoxicity). If the viability is lesser than 70% of the negative control, it shall be considered as cytotoxic potential.


Miller, F., Hinze, U., Chichkov, B., Leibold, W., Lenarz, T., & Paasche, G. (2017). Validation of eGFP fluorescence intensity for testing in vitro cytotoxicity according to ISO 10993‐5. Journal of Biomedical Materials Research Part B: Applied Biomaterials105(4), 715-722.

Prochor, P., & Mierzejewska, Ż. A. (2019). Influence of the surface roughness of PEEK GRF30 and Ti6Al4V SLM on the viability of primary human osteoblasts determined by the MTT test. Materials12(24), 4189.

Rose, E. C., Bumann, J., Jonas, I. E., & Kappert, H. F. (2000). Contribution to the biological assessment of orthodontic acrylic materials measurement of their residual monomer output and cytotoxicity. Journal of Orofacial Orthopedics/Fortschritte der Kieferorthopädie61(4), 246-257.

Meriç, G., Dahl, J. E., & Ruyter, I. E. (2008). Cytotoxicity of silica–glass fiber reinforced composites. Dental Materials24(9), 1201-1206.

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