Cell Culture Calculator
Cell counting, seeding, split ratio, and viability calculations.
Formula: cells/mL = (count ÷ squares) × dilution × 10⁴
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What it does
The Cell Culture Calculator handles the everyday arithmetic of mammalian cell culture: counting cells with a hemocytometer, computing the suspension volume needed to seed a target number of cells, deriving a split ratio for passaging, and measuring viability by Trypan blue exclusion. It is used by cell biologists, biotech technicians, and anyone maintaining adherent or suspension cell lines.
How it works
The hemocytometer formula is cells/mL = (count divided by squares) times dilution times 10 to the 4th power, where the 10 to the 4th factor converts one counting square of 0.1 cubic millimeters to one milliliter. Seeding volume equals target cells divided by stock concentration in cells per milliliter. The split ratio is defined by desired density divided by current density, so a current confluence of 90 percent diluted to a reseed density of 30 percent gives a 3 to 1 split; the rule is to keep 1 part cells and add (ratio minus 1) parts fresh medium. Viability equals live divided by (live plus dead) times 100 percent from Trypan blue counts.
Worked example
You count 250 cells across 4 hemocytometer squares from a 1 to 1 Trypan blue dilution, so the dilution factor is 2. Concentration equals (250 divided by 4) times 2 times 10 to the 4th power, which is 1.25 times 10 to the 6th cells per milliliter. To seed 5 times 10 to the 5th cells into a well, stock volume equals 5 times 10 to the 5th divided by 1.25 times 10 to the 6th, or 0.4 milliliters. If the flask is at 90 percent confluence and you want 30 percent after passaging, split ratio equals 90 divided by 30, or 3, so keep one third of the cells and add 2 volumes of medium.
When to use it
A researcher uses it to standardize seeding across 96 well replicates before a drug screen, to calculate how much medium to add when passaging a confluent T-75 flask, and to confirm a thawed vial is healthy by checking viability above 90 percent before starting an experiment.
FAQ
- What seeding density should I use for HEK293 cells?
- HEK293 typically seeds at 2 times 10 to the 5th to 3 times 10 to the 5th cells per square centimeter for rapid growth, but for transfection most protocols plate at 1 times 10 to the 5th to 1.5 times 10 to the 5th cells per square centimeter the day before to reach 70 to 90 percent confluence. Use the seeding volume mode with your stock concentration and target cell number.
- How do I calculate cell concentration from a hemocytometer?
- Count the cells in the corner and center squares, then apply cells per milliliter equals (count divided by number of squares) times dilution factor times 10 to the 4th power. This accounts for the 0.1 cubic millimeter volume of a counting square and the dilution you used.
- How do I calculate the split ratio when passaging cells?
- Divide the current confluence or density by the desired reseed confluence. For example 90 percent to 30 percent is a 3 to 1 split; keep 1 part of the cell suspension and add 2 parts fresh medium. The calculator applies split ratio equals current density divided by desired density.
- How do I calculate cell viability with Trypan blue?
- Mix cells 1 to 1 with Trypan blue, giving a dilution factor of 2, count live unstained and dead stained cells, then viability equals live divided by (live plus dead) times 100 percent. A healthy suspension is usually above 90 percent.