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Cell Seeding Calculator (Cells per Well and Seeding Density)

Enter your cell count and how many cells you want per well (or a density in cells/cm²), pick the plate and the number of wells, and get the volume of cell suspension and medium to add.

Inputs

cells/mL

Type 1e6 for 1×10⁶

Target per well

cells
mL

Results

Total cells
1.32×10⁶
Cell suspension to take
1.32 mL
Medium to add
11.88 mL
Total volume
13.20 mL

Common plate formats

PlateArea per wellTypical volume per well
6-well9.5 cm²2 mL
12-well3.8 cm²1 mL
24-well1.9 cm²0.5 mL
48-well0.95 cm²0.3 mL
96-well0.32 cm²0.1 mL

Typical values for Corning flat-bottom culture plates. Brands differ slightly, so check your datasheet when precision matters. Choosing a plate fills in its full well count and typical volume.

Formula

Cells per well = Density (cells/cm²) × Well area (cm²)

Suspension (mL) = Cells per well × Wells × 1.1 ÷ Conc. (cells/mL)

Medium (mL) = Volume per well (mL) × Wells × 1.1 − Suspension

Tip: Without the 10% extra, drop the × 1.1. You can type 1e6 for 1×10⁶ cells/mL.

You got this step right, but a whole experiment has dozens of linked parameters. Change one dilution factor and everything downstream has to be recalculated. In Vara the parameters of your whole experiment are connected, so changing one recalculates the rest automatically. Manage 3 experiments for free.

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How the formula works

  1. Start with the number of cells each well needs. If your protocol gives cells per well, use it directly. If it gives a seeding density in cells/cm², multiply by the well's growth area: cells per well = density × well area. This page uses 9.5 cm² for 6-well, 3.8 for 12-well, 1.9 for 24-well, 0.95 for 48-well and 0.32 cm² for 96-well plates.
  2. Work out how many wells to prepare for. For N wells with the 10% extra ticked, prepare for N × 1.1 wells; without it, prepare for N.
  3. Total cells = cells per well × wells prepared for.
  4. Suspension volume = total cells ÷ suspension concentration (cells/mL). Use the viable cell concentration from your count.
  5. Total volume = medium volume per well × wells prepared for, and medium to add = total volume − suspension volume. Mix the suspension and medium in one tube and dispense the same volume into every well; each well then gets the target number of cells.
  6. If the suspension volume comes out larger than the total volume, the suspension is too dilute to reach the target by adding medium. Spin the cells down, resuspend in less medium, count again and recalculate.

Worked examples

96-well plate: 5,000 cells per well in the inner 60 wells

  1. Given: viable cell concentration 1×10⁶ cells/mL, 5,000 cells per well, 100 μL (0.1 mL) per well, inner 60 wells, 10% extra ticked.
  2. Wells to prepare for: 60 × 1.1 = 66.
  3. Total cells: 5,000 × 66 = 330,000, or 3.3×10⁵.
  4. Suspension volume: 3.3×10⁵ ÷ 1×10⁶ cells/mL = 0.33 mL, which is 330 μL.
  5. Total volume: 0.1 mL × 66 = 6.6 mL. Medium to add: 6.6 − 0.33 = 6.27 mL.
  6. At the bench: add 330 μL of suspension to 6.27 mL of medium, mix, and dispense 100 μL per well. As a density that is 5,000 ÷ 0.32 = 15,625 cells/cm², which you can reuse when you switch plate formats.

6-well plate: a full plate at 2×10⁴ cells/cm²

  1. Given: viable cell concentration 1×10⁶ cells/mL, density 2×10⁴ cells/cm², 2 mL per well, 6 wells, 10% extra ticked.
  2. Cells per well: 2×10⁴ × 9.5 cm² = 1.9×10⁵.
  3. Wells to prepare for: 6 × 1.1 = 6.6.
  4. Total cells: 1.9×10⁵ × 6.6 = 1.254×10⁶.
  5. Suspension volume: 1.254×10⁶ ÷ 1×10⁶ cells/mL = 1.254 mL.
  6. Total volume: 2 mL × 6.6 = 13.2 mL. Medium to add: 13.2 − 1.254 = 11.946 mL.
  7. The calculator rounds volumes to two decimal places, so it shows 1.25 mL of suspension, 11.95 mL of medium and 13.20 mL in total; cell counts are shown to 3 significant figures, so the total reads 1.25×10⁶. Mix and dispense 2 mL per well.

Common mistakes

Reusing last time's suspension volume

You took 330 μL last time, so you take 330 μL again. Every passage gives a different count, so the suspension volume has to be recalculated from today's concentration, and the medium volume changes with it.

Adding the 10% extra to the medium only

Making extra medium without extra cells leaves every well short of cells. The extra has to apply to both cells and medium, which is what the wells-prepared-for number on this page does, so the concentration stays the same. Skipping the extra entirely is the other common problem: the last few wells run dry.

Using the total count instead of the viable count

At 80% viability, seeding from the total count puts only about 80% of the planned live cells in each well. Use the viable concentration from trypan blue or your automated counter.

Forgetting the dilution factor when counting

One large square of a hemocytometer holds 0.1 μL, so the mean count per square × 10⁴ × dilution factor = cells/mL. If you mixed the cells 1:1 with trypan blue, multiply by 2 as well; missing it throws every downstream number off by a factor of two.

Not remixing while you plate

Cells settle within minutes, so the first and last wells end up with different numbers. Gently pipette or invert the suspension every few columns. After plating, slide the plate back and forth and side to side rather than swirling it, or the cells pile up in the center or around the edge of each well.

Ignoring the edge effect

Outer wells lose more medium to evaporation and see bigger temperature swings, so growth and readouts often differ from the inner wells. Letting the plate sit at room temperature for a while before it goes into the incubator, and filling the outer wells with PBS or medium instead of samples, are common ways to handle it. Follow your protocol.

FAQ

How much suspension do I need for 5×10⁴ cells per well in a 24-well plate at 1×10⁶ cells/mL?+
With 10% extra, plan for 26.4 wells: 5×10⁴ × 26.4 = 1.32×10⁶ cells, so 1.32×10⁶ ÷ 1×10⁶ cells/mL = 1.32 mL of suspension. At 0.5 mL per well the total is 13.2 mL, so add 11.88 mL of medium, mix, and dispense 0.5 mL into each well.
How do I convert a seeding density (cells/cm²) into cells per well?+
Cells per well = density × well area. A 96-well plate well is about 0.32 cm², so at 1×10⁴ cells/cm² each well gets 3200 cells. Seeding by density keeps confluence consistent when you switch plate formats.
What is the growth area of each plate format?+
Typical values: 6-well about 9.5 cm², 12-well about 3.8 cm², 24-well about 1.9 cm², 48-well about 0.95 cm² and 96-well about 0.32 cm². Brands differ slightly, so check your plate's datasheet when precision matters.
Why add 10% extra?+
Liquid clings to tube walls and tips, so the last few wells often come up short. Preparing 1.1 times what you need makes sure every well gets its full amount. If your cells are scarce, untick the option and prepare for the exact number of wells.
What if the suspension volume comes out larger than the total volume?+
Your suspension is too dilute to reach the target by dilution alone. Spin the cells down, resuspend them in a smaller volume of medium, count again and recalculate.
Should I use the viable cell count?+
Yes. Trypan blue and most automated counters report both total and viable cells. Seed based on viable cells so each well gets the number of healthy cells you planned for.
How many cells should I seed per well in a 96-well plate?+
There is no single right number. It depends on the cell type, how fast it grows, the assay and when you read it, so go by your protocol or the paper you are following. Once you have a number, converting through density is the safest route: cells per well = density (cells/cm²) × 0.32 cm². If you are unsure, many labs seed a short range of densities in a pilot plate first and pick from that.
What is the formula for cell seeding?+
Four steps: cells per well = density × well area (skip this if your protocol already gives cells per well); total cells = cells per well × wells × 1.1 (10% extra); suspension volume = total cells ÷ suspension concentration; medium to add = volume per well × wells × 1.1 − suspension volume.
How do I scale cells per well from a 6-well to a 96-well plate?+
Scale by growth area so the seeding density stays the same. A 96-well well is about 0.32 cm² and a 6-well well about 9.5 cm². If you seed 2×10⁵ cells per 6-well well, the 96-well equivalent is 2×10⁵ × 0.32 ÷ 9.5 ≈ 6737 cells. Scale the medium volume to the plate format too (this page defaults to 2 mL for 6-well and 0.1 mL for 96-well), following your protocol.

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