Seeding density is per area
Adherent cells are usually seeded at a density in cells per cm² of growth area, so the same density gives the same coverage in any vessel. Typical growth areas:
- 96-well plate: 0.32 cm² per well
- 24-well plate: 1.9 cm² per well
- 12-well plate: 3.8 cm² per well
- 6-well plate: 9.5 cm² per well
- T25, T75 and T175 flasks: 25, 75 and 175 cm²
Check the manufacturer's sheet; areas differ slightly between brands.
Three steps
- Cells needed per vessel = density × area. At 20,000 cells/cm² in a 6-well plate: 20,000 × 9.5 = 190,000 cells per well.
- Total cells = cells per vessel × number of vessels, plus about 10% for pipetting losses. Six wells: 190,000 × 6 × 1.1 = 1,254,000 cells.
- Volume of suspension = total cells ÷ your count in cells/mL. With 1,000,000 cells/mL from the hemocytometer: 1.25 mL, made up with medium to the total volume you need (6 × 2 mL = 12 mL, plus 10%).
Picking the density
Density depends on the cell line and on when you want to use the cells. To reach a target confluence on a given day, work backwards from the doubling time: cells double once per doubling time once they recover from passaging, usually after about a day. A line that doubles every 24 hours seeded at 25% coverage is close to confluent after two to three days. Supplier datasheets and published protocols give a starting density for most lines.
Getting an even layer
Mix the suspension just before each transfer, as cells settle within a minute or two. After seeding, move plates in a cross pattern (forward-back, left-right) rather than swirling, which pushes cells to the edge of each well, and let them settle for 15 to 30 minutes before moving them to the incubator.
Tools
Count cells with the Hemocytometer Calculator, then enter the count and density in the Cell Seeding Calculator to get the suspension and medium volumes for plates and flasks. The Cell Doubling Time Calculator works out the doubling time from two counts.