Cell culture
Cell seeding calculator
Pick the plate or flask, enter your cell concentration and the seeding density, and get the number of cells per well, how much cell suspension and medium to mix, and what goes into each well. Growth areas are filled in for common vessels and stay editable, because they differ a little between manufacturers.
How the seeding calculation works
cells per well = density × area · suspension = cells per well × wells ÷ concentration · medium = volume per well × wells − suspension
Worked example: seeding a 96-well plate at 20,000 cells/cm² from a 1 × 10⁶ cells/mL suspension, 100 µL per well, with 10 % extra. Each well is 0.32 cm², so 6,400 cells per well. 96 wells × 1.1 = 105.6 wells' worth: 675,840 cells, which is 0.676 mL of suspension. The total volume is 10.56 mL, so add 9.88 mL of medium, mix, and put 100 µL in each well.
If the suspension alone is more than the final volume, the stock is too dilute for that density: spin the cells down, resuspend in less medium, and count again.
Growth areas and typical volumes
| Vessel | Growth area | Medium | Typical seeding (HeLa) |
|---|---|---|---|
| 6-well plate | 9.6 cm² | 1–3 mL | 0.3 × 10⁶ per well |
| 12-well plate | 3.5 cm² | 1–2 mL | 0.1 × 10⁶ per well |
| 24-well plate | 1.9 cm² | 0.5–1 mL | 0.05 × 10⁶ per well |
| 48-well plate | 1.1 cm² | 0.2–0.4 mL | 0.03 × 10⁶ per well |
| 96-well plate | 0.32 cm² | 0.1–0.2 mL | 0.01 × 10⁶ per well |
| 384-well plate | 0.084 cm² | ~0.08 mL | 0.0018 × 10⁶ per well |
| 35 / 60 / 100 mm dish | 8.8 / 21.5 / 56.7 cm² | 2 / 5 / 12 mL | 0.3 / 0.8 / 2.2 × 10⁶ |
| T-25 / T-75 / T-175 flask | 25 / 75 / 175 cm² | 3–5 / 8–15 / 35–53 mL | 0.7 / 2.1 / 4.9 × 10⁶ |
Values from Thermo Fisher's cell culture reference table (Nunc vessels). Other brands differ slightly, for example a Corning 6-well is 9.5 cm²; the area field above is editable. The right density depends on the cell line and on when you want the culture to reach confluence, so treat the HeLa numbers as a starting point.
Seeding evenly
- Count with a hemocytometer or automated counter just before seeding, and use the live-cell concentration.
- Keep the mix in suspension: invert or pipette it between rows, because cells settle within minutes.
- Let a freshly seeded plate sit flat at room temperature for 15–30 minutes before the incubator to reduce edge crowding.
- For imaging, image a few fields per well and check density on the images: with a calibrated image, cells per mm² comes straight from a count.
Questions
How do I calculate cells per well?
Multiply the seeding density in cells per cm² by the growth area of one well. A 96-well plate well is about 0.32 cm², so 20,000 cells/cm² is 6,400 cells per well.
How much cell suspension do I need?
Total cells needed ÷ cell concentration. For 96 wells of 6,400 cells (plus 10 % extra) from 1 × 10⁶ cells/mL, that is 675,840 ÷ 1,000,000 ≈ 0.68 mL, topped up with medium to the final volume.
Is this the same as C1V1 = C2V2?
Yes. The calculator solves C1 × V1 = C2 × V2 for cells: C1 is your stock concentration, C2 the concentration of the seeding mix (cells per well ÷ volume per well), V2 the total volume, and V1 the suspension to take.
What is the surface area of a 96-well plate well?
About 0.32 cm² for a standard flat-bottom well (Thermo Fisher and Corning both list 0.32 cm²); half-area 96-well plates are about 0.16 cm².
Is this calculator free?
Yes. It runs in your browser and sends nothing anywhere. SlideScope, the desktop microscopy app made by the same team, is a paid app with a free preview.
Sources
Results are theoretical or arithmetic; check them against your instrument's documentation before relying on them for clinical or regulated work.
Measure on your real images
SlideScope opens 34 microscope and slide-scanner formats, CZI, ND2, SVS and LIF among them, with their own calibration, so lengths, areas, cell counts and scale bars come out in µm without setting anything up. Open a few of your own images with the free preview. Already decided? Subscribe now.