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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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.

Do not store MTT solution at 4°C for more than a few days.

Prepare MTT solvent 4 mM HCl, 0.1% NP40 in isopropanol.

Stage 2 - Assay protocol

Steps

Discard media from cell cultures.

An alternative method is to add an equal volume of MTT solution to the existing media in the culture. Ensure that the same volume of existing media is present for each sample.

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.

Occasionally, pipetting of the liquid may be required to fully dissolve the MTT formazan.

Read absorbance at OD=590 nm.

Read plate within 1 hour.

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.

The amount of absorbance is proportional to cell number.

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))