MTT Assay / IC50 Calculator
Calculate IC50, cell viability percentage, and dose-response curve parameters from MTT assay data.
What MTT Actually Measures
MTT is reduced to purple formazan by cellular reductases, mainly NAD(P)H-dependent. The signal therefore reflects metabolic activity, not cell number directly — a distinction that matters more often than it is acknowledged.
| Situation | Effect on MTT signal |
|---|---|
| Metabolic inhibition without death | Falsely low viability |
| Compound stimulates metabolism | Falsely high — can exceed 100% |
| Cell cycle arrest, cells alive | Reduced signal, no death |
| Compound reduces MTT chemically | False signal — ascorbate, some polyphenols |
Directly reducing compounds are the most dangerous artefact, because they generate formazan without any cells present. A cell-free control containing compound and MTT detects this and should be routine for any novel chemistry.
Why IC50 Is Not a Constant
IC50 depends on assay duration, seeding density, and readout. A 72-hour exposure gives a lower IC50 than 24 hours for most cytotoxics, and denser cultures shift it upward.
| Variable | Effect on IC50 |
|---|---|
| Longer exposure | Lowers |
| Higher seeding density | Raises |
| Serum concentration | Raises for protein-bound drugs |
| Readout method | Shifts systematically |
IC50 values are therefore not comparable across protocols, and publications should state duration, density and assay. Comparisons within a single experiment are meaningful; comparisons against literature values often are not.
Fitting the Curve
IC50 should be obtained from a four-parameter logistic fit across concentrations spanning the full response, ideally with the top and bottom plateaus defined. Interpolating between two points near 50% assumes a slope the data has not established.
A Hill slope well above 1 suggests cooperative action or a threshold effect; well below 1 suggests a heterogeneous population or multiple targets. The slope carries information and should be reported alongside the IC50.
Worked Examples
Common Mistakes
It measures metabolic reductase activity. Cells arrested but alive give reduced signal without any death occurring.
Ascorbate and various polyphenols reduce MTT directly, generating formazan with no cells present. This control is essential for novel chemistry.
IC50 depends on exposure duration, seeding density and serum concentration. Literature comparisons require matched conditions.
A proper four-parameter fit across the full concentration range is needed. Two-point interpolation assumes a slope the data has not established.