Cell culture
Trypan blue viability
From live and dead counts under trypan blue, this gives per-cent viability, the concentration of live, dead and total cells, and how much suspension to take for a target number of live cells. Dead cells take up the dye and appear blue; live cells exclude it and stay clear.
Viable-cell yield
Viability is live cells over the total. Concentrations use the same haemocytometer arithmetic as the counting tool: mean cells per square times the dilution factor times 10⁴. There is no universal minimum viability — what counts as acceptable depends on the line and the downstream use — so this tool reports the figure rather than judging it. Read the chamber within a few minutes of adding trypan blue, as prolonged exposure kills cells and lowers the apparent viability.
Formula
viability = live ÷ (live + dead) × 100
- live
- clear, unstained cells counted
- dead
- blue, dye-filled cells counted
- live concentration
- (live ÷ squares) × dilution factor × 10⁴
Worked example
A count of 184 clear and 16 blue
- 184 live, 16 dead over 4 squares
- dilution factor 2
- suspension volume 10 ml
- Viability: 184 over 200 is 92 per cent.
- Live concentration: 184 over 4, times 2, times 10⁴, is 9.2 × 10⁵ per ml.
- Total live in 10 ml: 9.2 × 10⁶.
Answer 92 per cent viable, 9.2 × 10⁵ live cells per ml
Common mistakes
- Leaving the cells in trypan blue too long. The dye is toxic; read within a few minutes or viability drifts down for reasons that have nothing to do with your treatment.
- Treating a fixed per-cent as pass or fail. What counts as acceptable depends on the line and the assay ahead; a primary isolate and a robust cell line do not share a threshold.
- Scoring faint cells inconsistently. Decide before you start whether a pale cell is live or dead, and apply it the same way across every square and every sample.
Frequently asked
Is trypan blue reliable near the low end?
It reports membrane integrity, not metabolic health, and undercounts early apoptosis. For a viability that must be defended, confirm with a second method.
Do I count dead cells for seeding?
No. Seed on the live concentration; that is why this tool separates it out and works the volume from live cells rather than total.
Why did total concentration fall after treatment?
Dead cells lyse and disappear, so a harsh treatment can lower both viability and the total count at once. Compare against a control counted the same way.