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Cell Viability Calculator (Trypan Blue)

Calculate percent viability from a trypan blue exclusion count of live (unstained) and dead (stained) cells, with the uncertainty of the count.

Formula

viability %=nlivenlive+ndead×100\text{viability}\,\% = \dfrac{n_{\text{live}}}{n_{\text{live}} + n_{\text{dead}}}\times 100
nliven_{\text{live}}
cells that exclude the dye and appear clear
ndeadn_{\text{dead}}
cells that take up the dye and appear blue

How it works

Trypan blue cannot cross an intact cell membrane. Cells with a damaged membrane take up the dye and stain blue, while live cells remain clear. Viability is the share of all counted cells that are unstained.

The percentage inherits the sampling error of the count, so counting more cells gives a tighter estimate.

Worked example

186 unstained and 14 blue cells are counted.

  1. Total = 186 + 14 = 200.
  2. Viability = 186 / 200 × 100 = 93.0%.

Viability is 93.0%, from a count of 200 cells.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Membrane integrity is used as a stand-in for viability. Cells early in apoptosis still exclude the dye.
  • Cells are counted within a few minutes of adding the dye.

Common mistakes

  • Leaving cells in trypan blue too long. The dye is toxic, and viable cells begin to take it up after several minutes.
  • Staining in medium containing serum, where proteins bind the dye and darken the background.
  • Reporting a percentage from a few dozen cells as if it were precise.

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.