—
%
2,000,000
cells/mL
1,700,000
cells/mL
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%
2,000,000
cells/mL
1,700,000
cells/mL
The Trypan Blue Exclusion Calculator determines cell viability and concentration using the trypan blue dye exclusion method. Trypan blue is a vital stain that cannot penetrate intact cell membranes. Live cells with intact membranes exclude the dye and appear clear (unstained), while dead cells with compromised membranes absorb the dye and appear blue.
This is one of the most widely used methods in cell culture laboratories for assessing cell health before experiments, passaging, or cryopreservation. Results are typically obtained using a hemocytometer counting chamber.
The calculator uses standard hemocytometer counting formulas:
The factor of 10,000 (10⁴) converts the count from a hemocytometer square (0.1 µL volume) to cells per milliliter. The dilution factor accounts for mixing with trypan blue (typically 1:1, giving a factor of 2).
Inputs
Results
85% viability with 1.7 × 10⁶ viable cells/mL. Viability above 80% is generally acceptable for most cell culture applications.
Inputs
Results
45% viability after thawing from cryopreservation is poor but not uncommon. Recovery culture may improve viability over 24-48 hours.
For most cell culture experiments, viability above 90% is considered excellent, 80-90% is acceptable, and below 80% may indicate problems with culture conditions. For cryopreserved cells, post-thaw viability of 70-80% is typical. Specific thresholds depend on the cell type and intended application.
The standard hemocytometer counting area has a volume of 0.1 µL (0.0001 mL). Multiplying the cell count by 10,000 converts the count to cells per milliliter. This conversion factor is specific to the standard Neubauer hemocytometer chamber dimensions.
Trypan blue only distinguishes cells with intact vs. compromised membranes. It cannot detect early apoptosis, functional impairment, or distinguish between viable but non-proliferative cells. The dye is also toxic with prolonged exposure, so counting must be done within 3-5 minutes of staining. For more sensitive viability assessment, flow cytometry with annexin V/PI staining is preferred.
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