How do you calculate cell viability with trypan blue?
Trypan blue enters cells with damaged membranes, so dead cells stain blue and live cells stay clear. Viability (%) = live cells ÷ (live + dead cells) x 100 (Strober, 2015). Mix the cells 1:1 with 0.4% trypan blue, which gives a dilution factor of 2, and count within 3 to 5 minutes, because longer exposure kills cells.
How do you calculate cells per mL from a hemocytometer?
Cells per mL = average count per large square x dilution factor x 10⁴. The 10⁴ comes from the volume over one large square of a standard hemocytometer: 1 mm x 1 mm x 0.1 mm = 0.1 µL, or 10⁻⁴ mL.
Worked example: 180 live and 20 dead cells across 4 large squares, mixed 1:1 with trypan blue. Viability = 180 ÷ 200 x 100 = 90%. Live cells/mL = 180 ÷ 4 x 2 x 10⁴ = 900,000. In a 10 mL suspension that is 9 million live cells.
How many cells should you count?
Counting error falls as the count rises, so count enough squares to reach at least about 100 cells; the calculator warns below that. Count cells touching two agreed borders of each square and not the other two, so no cell is counted twice. Next, use the live-cell concentration in the Cell Seeding Density Calculator.
Frequently asked questions
What is the formula for cell viability?
Viability (%) = live cells ÷ total cells x 100, where total = live + dead.
What is the dilution factor for trypan blue?
2 when the cells are mixed 1:1 with trypan blue. If you also diluted the cells beforehand, multiply the factors.
Why multiply by 10⁴?
Each large square of a standard hemocytometer holds 0.1 µL (1 mm x 1 mm x 0.1 mm), which is 10⁻⁴ mL, so the count per square times 10⁴ gives cells per mL.
How long can cells sit in trypan blue?
Count within 3 to 5 minutes. Longer exposure starts to kill cells and lowers the apparent viability.
Does this work for automated cell counters?
Automated counters report viability and concentration directly. This calculator is for manual hemocytometer counts.
Is anything I enter saved or sent?
No. The calculator runs in your browser. Nothing you type is saved, sent to BioBridgeX or added to the page address, and analytics never see your numbers.
Related free tools
- Cell Seeding Density Calculator: Cells per well or per cm² into the cell suspension and medium to mix for a plate, with spare volume.
- In Vitro Assay and Screening Planner: Assay type, cell models, compounds, concentration series, controls, replicates and readout as an organized assay plan.
- All lab calculators and every free tool on BioBridgeX.
Sources
- Strober W. Trypan blue exclusion test of cell viability. Current Protocols in Immunology 2015;111:A3.B.1-A3.B.3
- Thermo Fisher Gibco Cell Culture Basics: Trypan blue exclusion
Check every result before use. Formulas assume ideal conditions; follow your validated lab procedures and the reagent supplier's instructions. Facts on this page last checked 27 October 2026.
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