Cell Seeding Calculator
Pick the plate, dish or flask, set the density you want as cells per cm² or cells per well, and enter the concentration of your cell suspension. The calculator returns the volume of suspension and fresh medium for the whole seeding mix, how much to put in each well, and the total number of cells you need. Runs entirely in your browser. Free, no sign-up.
Use your vessel maker's figure if it differs from the table.
From your cell count, for example a hemocytometer.
Cells plus medium once seeded.
Covers pipetting loss; 10% is common.
Results
0.5016
Cell suspension to take (mL)
12.7
Fresh medium to add (mL)
38,000
Cells per well or vessel
38
Suspension per well or vessel (µL)
501,600
Total cells needed
20,000
Density reached (cells/cm²)
Seeding mix
| Component | Volume (mL) |
|---|---|
| Cell suspension | 0.5016 |
| Fresh medium | 12.7 |
| Total seeding mix | 13.2 |
Mix gently, then dispense 1 mL into each of 12 wells or vessels.
The working
cells per vessel = 20,000 cells/cm² × 1.9 cm² = 38,000
suspension = cells needed ÷ concentration = 501,600 ÷ 1 × 10⁶ = 0.5016 mL
How to Use the Cell Seeding Calculator
- 1. Choose the vessel. The growth area comes from the table in the calculator. If your plate's area differs, type it into the override box.
- 2. Set the density. Enter cells per cm² (useful when you move between formats) or cells per well, which is how many protocols are written. The button next to the vessel fills in Thermo Fisher's starting number.
- 3. Enter the suspension. Use the concentration from your count, and the total volume you want in each well once the cells are in. Add extra mix to cover pipetting losses.
- 4. Make the seeding mix. Combine the volumes shown for the suspension and the fresh medium, mix gently, and dispense the same amount into every well.
The Arithmetic
Cells per well
cells per well = density (cells/cm²) × growth area (cm²)
Suspension
volume of suspension (mL) = total cells needed ÷ cells/mL in the suspension
Medium
fresh medium = volume of the seeding mix − suspension
The seeding mix is made in one tube for all the wells, for N × (1 + extra) wells so the last one is not short, and the suspension makes up only a small part of it. Making one mix and dispensing it, rather than adding cells to each well individually, is quicker and gives much more uniform wells. Count the suspension first (a Hemocytometer Cell Count Calculator turns square counts into cells/mL), and resuspend well before every step because cells settle quickly.
Growth Areas and Starting Numbers
The calculator's vessel table is taken from Thermo Fisher Scientific's Useful Numbers for Cell Culture (Gibco Cell Culture Basics), where the starting numbers were measured with HeLa cells. Representative values:
| Vessel | Growth area (cm²) | Starting number | Growth medium (mL) |
|---|---|---|---|
| 6-well plate | 9.6 | 0.3 × 10⁶ per well | 1–3 |
| 12-well plate | 3.5 | 0.1 × 10⁶ per well | 1–2 |
| 24-well plate | 1.9 | 0.05 × 10⁶ per well | 0.5–1.0 |
| 96-well plate | 0.32 | 0.01 × 10⁶ per well | 0.1–0.2 |
| 100 mm dish | 56.7 | 2.2 × 10⁶ | 12 |
| T-75 flask | 75 | 2.1 × 10⁶ | 8–15 |
Treat the starting numbers as a place to begin, not a rule: the right density depends on the cell type, how long the experiment runs and how fast the cells divide. Growth areas also differ a little between manufacturers, so check the product sheet and override the area when your vessel's figure is different. Corning's guidance of 0.2–0.3 mL of medium per cm² is a useful cross-check for flasks and dishes, though it does not extend to the small wells of microplates, where the meniscus changes the ratio.
Worked Example: Twelve Wells of a 24-Well Plate
You want 20,000 cells/cm² in twelve wells of a 24-well plate, each filled to 1 mL, from a suspension at 1 × 10⁶ cells/mL, with 10% extra mix (the calculator's defaults).
- Cells per well: 20,000 × 1.9 = 38,000, which is 38 µL of suspension per well.
- Mix for 12 × 1.1 = 13.2 wells: 13.2 mL in total containing 38,000 × 13.2 = 501,600 cells.
- Suspension: 501,600 ÷ 1,000,000 = 0.5016 mL. Fresh medium: 13.2 − 0.5016 = 12.7 mL.
- Dispense 1 mL of the mix into each well.
When the Suspension Is Too Dilute
If your cells are at 10,000 cells/mL, getting 38,000 into a well needs 3.8 mL of suspension, more than the 1 mL the well holds. The calculator flags this instead of reporting a negative amount of medium. Spin the cells down at a gentle speed (the Centrifuge RPM to RCF Calculator converts a protocol's × g to the rpm for your rotor), discard the supernatant and resuspend in a smaller volume, then count again before you seed.
Common Mistakes
- Counting dead cells. Seed on the live-cell count, and use the viability from the trypan blue count.
- Mixing the units. Cells per cm² and cells per well are 1.9 times apart in a 24-well plate. Check which one the protocol means.
- Not remixing. Cells sink within minutes, so a pipette taken from the top of an unmixed tube is low in cells.
- Seeding the edge wells. Evaporation in the outer wells of a plate changes concentrations; many labs fill them with buffer or medium and leave them unseeded.
- Forgetting passage and cell type. A density that suits fast-growing HeLa cells will overgrow slower or contact-inhibited lines.
Frequently Asked Questions
- How do you calculate how many cells to seed per well?
- Multiply the target density in cells/cm² by the well's growth area in cm². A 24-well plate well has about 1.9 cm², so 20,000 cells/cm² means 38,000 cells per well. If your protocol gives cells per well directly, enter that.
- How do you calculate the volume of cell suspension to add?
- Divide the number of cells you need by the cells per millilitre in your suspension. 38,000 cells from a 1 × 10⁶ cells/mL suspension is 0.038 mL, or 38 µL. For many wells, make one seeding mix and divide it.
- How many cells should I seed in a 24-well plate?
- Thermo Fisher's table suggests about 50,000 cells per well for HeLa cells, which is about 26,000 cells/cm². The right number depends on the cell line and how long the experiment runs: use fewer cells for a longer culture or fast growers, and more if the cells must be confluent the next day.
- How many cells are in a confluent T-75 flask?
- Thermo Fisher lists about 8.4 × 10⁶ HeLa cells at confluency in a T-75 flask (75 cm²), roughly 112,000 cells/cm². This varies a lot with cell type and size, so use it only as a rough guide.
- Why add extra volume to the seeding mix?
- Liquid is lost to the walls of the tube and the pipette tip, so a mix made for exactly N wells leaves the last well short. 10% extra is typical; with only a few wells, make the mix for one extra well instead.
- Does the medium volume change the number of cells?
- No. The number of cells per well is set by the density and the area. The volume you seed in only changes how dilute the cells are, and how much of that volume the suspension takes up.