Hemocytometer cell count and cell seeding calculator
Cells per mL = the mean count per large corner square × 10,000 × the dilution factor; with equal volumes of cells and trypan blue the dilution is 2. Viability is live cells ÷ (live + dead) × 100. The calculator then gives the volume of suspension and medium to seed a set number of cells in each well.
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At a glance
| Formula | cells/mL = mean per large square × 10,000 × dilution |
|---|---|
| Viability | live ÷ (live + dead) × 100 |
| Dilution | (cells + trypan blue) ÷ cells, times any earlier dilution |
| Seeding | By cells per well or by cells/cm² and growth area |
| Output | Cells/mL, viability, suspension and medium volumes; CSV |
| Runs in | Your web browser; nothing is uploaded |
| Price | Free, no account needed |
What it does
How to use the hemocytometer cell count calculator
- Mix and loadMix your cells with trypan blue (for example 10 µL + 10 µL), load the hemocytometer and count within a few minutes.
- Enter the countsType the live (clear) cell count of each large corner square, and the blue (dead) counts in the same order for the viability.
- Enter the dilutionType the volumes of cells and trypan blue you mixed, and any dilution made before that.
- Plan the seedingEnter cells per well (or cells/cm² and the growth area), the number of wells and the volume per well.
- Read and pipetteRead the volume of cell suspension and medium to mix, then add the stated volume to each well.
How it works
cells/mL = mean count per large square × 10,000 × dilution
dilution = (cell volume + dye volume) / cell volume × earlier dilution
viability (%) = live / (live + dead) × 100
In the hemocytometer protocol of Phan et al. (2022) the unstained cells in each large corner square (16 small squares) are counted, the counts are averaged, and the mean is multiplied by 10,000 and by the trypan blue dilution to give viable cells per mL of the original suspension. A cell is counted when it lies inside the square or on its right-hand or bottom line, so cells on the edges are not counted twice. The factor 10,000 means each large square holds 10⁻⁴ mL.
Trypan blue enters cells whose membrane is damaged, so live cells stay clear and dead cells turn blue (Strober 2015). In Strober's protocol equal parts of cell suspension and 0.4% trypan blue are mixed, a 2-fold dilution; Phan et al. mix 0.1 mL of cells with 0.4 mL of dye, a 5-fold dilution. Strober gives viability as viable cells per mL ÷ total cells per mL × 100, asks for counting within 3 to 5 minutes of mixing (longer incubation kills cells) and for serum-free solution, because serum proteins take up the dye.
For seeding, the cells needed are cells per well × number of wells (plus any extra), the volume of suspension is cells needed ÷ viable cells per mL, and the rest of the final volume is medium.
Sources: Phan A, Romeis E, Tantalo L, Giacani L. In vitro transformation and selection of Treponema pallidum subsp. pallidum. Curr Protoc 2022;2:e507 (Support Protocol 2), doi:10.1002/cpz1.507. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2015;111:A3.B.1–A3.B.3, doi:10.1002/0471142735.ima03bs111.
Worked example
Cells mixed 10 µL + 10 µL with trypan blue (dilution 2). The four large corner squares hold 48, 52, 45 and 51 clear cells (mean 49) and 3, 2, 4 and 3 blue cells (mean 3).
Viable cells: 49 × 10,000 × 2 = 980,000 cells/mL. Total cells: 52 × 10,000 × 2 = 1.04 × 10⁶ cells/mL. Viability: 49 / 52 × 100 = 94.2%.
To seed 20,000 cells in each of 24 wells in 0.5 mL: 480,000 cells are needed, which is 480,000 / 980,000 = 490 µL of the suspension, made up with 11.51 mL of medium to 12 mL (40,000 cells/mL); add 0.5 mL to each well.
Frequently asked questions
How do you calculate cells per mL from a hemocytometer?
Average the counts of the large corner squares, multiply by 10,000 and by the dilution factor. With a mean of 49 cells per square and cells mixed 1:1 with trypan blue, that is 49 × 10,000 × 2 = 980,000 cells/mL (Phan et al. 2022).
Why multiply by 10,000?
Each large corner square of the hemocytometer holds 10⁻⁴ mL, so the number of cells in one square times 10,000 is the number per mL of the mix you loaded.
What is the dilution factor with trypan blue?
The total volume divided by the volume of cells: equal volumes (for example 10 µL + 10 µL) give 2, and 0.1 mL of cells with 0.4 mL of dye gives 5. Multiply by any dilution you made before adding the dye.
How is cell viability calculated?
Viability (%) = live (unstained) cells ÷ total (unstained + blue) cells × 100 (Strober 2015). With 49 live and 3 dead cells per square, it is 94.2%.
Which cells on the lines should I count?
Count cells inside the square and those touching its right-hand or bottom line, and leave out those on the top and left lines, so no cell is counted twice (Phan et al. 2022).
How long can cells sit in trypan blue?
Count within 3 to 5 minutes of mixing; longer incubation kills cells and lowers the viability. Mix in serum-free solution, because serum proteins stain with trypan blue (Strober 2015).
How do I work out how much cell suspension to seed?
Multiply cells per well by the number of wells, divide by the viable cells per mL to get the volume of suspension, and make it up to the final volume (wells × volume per well) with medium.
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