MTT assay protocol
Detailed instructions on reagent preparation and assay protocol for an MTT assay to measure cell proliferation or cell cytoxicity.
Last edited Fri 23 Jul 2021
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- Ready-to-use reagents
- Colorimetric
- Validated on adherent cells and suspension cells
- Quantitative assay
- Results in 3h 30min
Stage 1 - Reagent preparation
Steps
MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and culture media (5 mg/mL). We recommend using a 5 mg/mL solution in PBS.
Prepare MTT solution.
- Mix by vortexing or sonication.
- Filter sterilize solution after adding MTT.
- Store MTT solution at -20°C (stable for at least 6 months).
Prepare MTT solvent 4 mM HCl, 0.1% NP40 in isopropanol.
Stage 2 - Assay protocol
- Serum or phenol red present in the culture medium can generate background. If your sample contains serum or phenol red, set up sample background controls: 50 µL MTT reagent + 50 µL cell culture media (no cells).
- Prepare parallel well(s) as solvent control and use same volume of solvent as for the treated cells.
Steps
Discard media from cell cultures.
- For adherent cells, carefully aspirate the media.
- For suspension cells, spin the 96 well plate at 1,000 xg, 4°C for 5 minutes in a microplate-compatible centrifuge and carefully aspirate the media.
Add 50 µL of serum-free media and 50 µL of MTT solution into each well.
Incubate the plate at 37°C for 3 hours.
After incubation, add 150 µL of MTT solvent into each well.
Wrap plate in foil and shake on an orbital shaker for 15 minutes.
Read absorbance at OD=590 nm.
Stage 3 - Data analysis
Steps
Average the duplicate reading for each sample.
Subtract the culture medium background from your assay reading. This is the corrected absorbance.
For cell counting, a standard curve can be established with known cell number and fixed incubation times with the assay reagent.
Average the duplicate reading for each sample.
Subtract the culture medium background from your assay readings. This is the corrected absorbance.
Calculate percentage cytotoxicity with the following equation, using corrected absorbance.
% cytoxicity = (100 x (control - sample))