How to Analyse MTT Assay Data: % Viability, IC50 and Statistics

The MTT assay measures cell viability: living cells reduce the yellow MTT dye to purple formazan, and absorbance (usually read around 570 nm) is proportional to the number of metabolically active cells. Here is how to turn raw readings into results.

1. Organise your plate data

Record absorbance for: blank (medium + MTT, no cells), control (untreated cells) and each treatment concentration, with replicates (commonly triplicates).

2. Blank correction

Subtract the mean blank absorbance from every well: Corrected OD = OD(sample) − mean OD(blank).

3. Percentage viability

% Viability = (mean corrected OD of treated / mean corrected OD of control) × 100

The control is therefore 100%. Percentage inhibition = 100 − % viability.

4. Mean and SD for each concentration

Calculate % viability for each replicate, then the mean and standard deviation for each concentration. Ideally repeat the whole experiment on separate days (biological replicates).

5. IC50

IC50 is the concentration that reduces viability to 50%. The best method is non-linear regression on a dose–response curve (log[concentration] vs % viability, four-parameter logistic model) in software such as GraphPad Prism. A rough estimate can come from linear interpolation between the two concentrations around 50%, but say which method you used.

6. Statistical comparison

• Several concentrations vs control → one-way ANOVA followed by Dunnett’s test (each group vs control) or Tukey’s test (all pairs).
• Only one treatment vs control → independent t-test.
• Mark significance on graphs: * p < .05, ** p < .01, *** p < .001.

7. Presenting results

A bar chart (concentration on the x-axis, % viability on the y-axis, mean ± SD error bars, significance stars) or a dose–response curve with the IC50 marked. Example sentence: “The extract reduced cell viability in a concentration-dependent manner, with an IC50 of 48.6 µg/mL (illustrative value).”

Common mistakes

• Not subtracting the blank.
• Calculating viability from single wells and ignoring replicates.
• Calling technical replicates “n = 3 experiments”.
• Reporting IC50 without the fitting method.

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