Analysis
MTT viability assay
Turn MTT absorbance readings into per-cent viability against a control, with a mean and spread per group. For a concentration series it interpolates an IC50 from the points that bracket fifty per cent. Enter the blank so the dye-and-plate background is removed.
One group per line: a label (or a concentration, in series mode), then one or more absorbance replicates separated by commas. In series mode the row with concentration 0 is the control.
The IC50 is interpolated on a log scale between the two concentrations that bracket 50 per-cent viability. It is a reading off your points, not a fitted dose-response curve, and appears only when viability actually crosses 50 per cent within the range tested.
Viability per group is the blank-subtracted absorbance over the blank-subtracted control, as a per-cent. Replicates give a mean and a standard deviation. The blank is the medium-plus-reagent absorbance with no cells; subtracting it removes the background the dye and plate contribute.
Formula
viability = (sample − blank) ÷ (control − blank) × 100
- sample
- the mean absorbance of the treated group
- control
- the untreated reference, taken as 100 per cent
- blank
- medium plus reagent, no cells; the background to subtract
- IC50
- interpolated on a log scale between the two doses that straddle 50 per cent
Worked example
A treated group against an untreated control
- control mean 1.00
- treated mean 0.525
- blank 0.05
- Subtract the blank: 0.525 minus 0.05 is 0.475; 1.00 minus 0.05 is 0.95.
- Divide: 0.475 over 0.95 is 0.50.
- As a per cent: 50.
Answer 50 per-cent viability relative to the control
Common mistakes
- Skipping the blank. Formazan sits on a background from medium, phenol red and the plate; without subtracting it, low-signal wells read too high.
- Reading an IC50 off a curve that never reaches half. If viability stays above fifty across your range, there is no IC50 to interpolate; extend the doses rather than extrapolating.
- Comparing plates read at different times or wavelengths. Formazan development and the read wavelength both shift absolute values; keep a control on every plate.
Frequently asked
Is the IC50 a fitted curve?
No. It is a straight interpolation between the two bracketing points on a log-dose scale. For a publication-grade value, fit a four-parameter model to all the points.
How many replicates?
Enough to see the spread; three technical wells per group is a common minimum, and the tool reports the standard deviation so you can judge it.
Can I use it for other tetrazolium assays?
Yes. MTS, XTT and WST share the same absorbance-to-viability arithmetic; only the reagent handling differs.