How to calculate IC50: viability and the 4PL dose–response curve
IC50 is the drug concentration that halves cell viability (or activity) — the smaller, the more potent. Convert absorbance to viability, fit an S-shaped curve (4PL) against log concentration, and read the curve’s midpoint.
Paste absorbance values — blanks are subtracted, viability calculated, and a 4PL fit gives IC50 with 95% CI and a comparison between drugs.
Open the IC50 calculator →1. Absorbance → viability (%)
The blank is a well with medium and reagent but no cells. Without blank subtraction, background absorbance inflates viability and the curve never reaches 0%.
2. The four 4PL parameters
| Parameter | Meaning | Typical |
|---|---|---|
| Top | Viability at very low concentration | about 100% |
| Bottom | Viability at very high concentration | near 0% (higher if killing is incomplete) |
| IC50 | Concentration halfway between top and bottom | — |
| Hill slope | How steeply the curve falls | 0.5–3 |
Always put log concentration on the x-axis. Diluting in 3- or 10-fold steps spaces the points evenly on that log scale.
3. 95% confidence intervals and relative vs absolute IC50
“IC50 = 0.80 µM (95% CI 0.75–0.86)” means the true IC50 very likely lies in that range. An interval wider than 10-fold means too few concentrations around the middle of the curve.
When the curve bottom is not 0% (e.g. it levels off at 20%), two IC50s differ: the relative IC50 is halfway between top and bottom (the value usually reported), and the absolute IC50 is where viability is exactly 50%. State which one you used in the Methods.
4. A good concentration design
- At least 6–8 concentrations in 3-fold (or half-log) steps, so that both the top and bottom plateaus are visible.
- Three or more wells per concentration and three or more independent experiments on different days; calculate the IC50 in each and average them.
- Keep the vehicle (DMSO, etc.) at the same concentration in every well and use vehicle-only wells as the 100% control.
5. Common mistakes
- Reporting an IC50 outside the tested range: if viability never drops below 50%, report “IC50 > highest concentration”.
- Reading IC50 by straight-line interpolation between the two points around 50% ignores the curve and is inaccurate.
- Edge effects: outer wells of a 96-well plate drift with evaporation. Fill the outer ring with PBS.
Paste absorbance values — blanks are subtracted, viability calculated, and a 4PL fit gives IC50 with 95% CI and a comparison between drugs.
Open the IC50 calculator →