Cell Dilution Calculator

How to use this calculator

Enter the live-cell density you counted (cells per mL), the target density from your protocol, and the final volume you need. The calculator returns the volume of suspension to pipette, the medium to add, and the total number of cells in the final suspension. Large numbers can be typed as 2e6 for 2,000,000.

Working per well? Enter the total volume for all wells plus a little extra (for a 24-well plate at 0.5 mL per well, 13 mL rather than 12 mL), or enter the total number of cells you need and let the calculator find the final volume at the target density.

The formula

N = C₂ × V₂ = C₁ × V₁

V_medium = V₂ − V₁

C₁: stock cell density
C₂: target cell density
V₂: final volume
V₁: cell suspension to pipette
V_medium: medium to add
N: total cells in final suspension

Worked example

After counting, your suspension holds 2 × 10⁶ cells/mL. You want 10 mL at 5 × 10⁵ cells/mL to seed a flask.

Cells needed = 5 × 10⁵ × 10 = 5 × 10⁶ cells.
Suspension volume = 5 × 10⁶ ÷ 2 × 10⁶ = 2.5 mL.
Medium = 10 − 2.5 = 7.5 mL.

Combine 2.5 mL of cell suspension with 7.5 mL of fresh medium.

Assumptions and limits

C₁ is the viable cell density. If your counter reports total cells, multiply by viability (e.g. 0.92 for 92% viable).
The suspension is evenly mixed when you pipette; resuspend gently right before taking the aliquot.

Frequently asked questions

How do I calculate cells/mL from a hemocytometer?

Average the count per large square, multiply by the dilution factor (2 for a 1:1 trypan blue mix) and by 10⁴. 50 cells per square × 2 × 10⁴ = 1 × 10⁶ cells/mL.

What if my stock is less concentrated than the target?

You cannot dilute upward. Centrifuge the cells, remove supernatant and resuspend in a smaller volume, then recount.

What is a typical seeding density?

It varies by cell line. Many adherent lines are seeded at 1–5 × 10⁴ cells/cm²; check the supplier’s data sheet.