How to use
- ① Choose the culture mode. Adherent takes the target in cells/cm², suspension in cells/mL. For plates, choose how they are filled as well.
- ② Enter the current cell density and viability. The density is what the cell counting calculator gives you. Nothing is shown until this box has a number in it.
- ③ Check the target seeding density and add vessel rows. Choosing a vessel brings the source's recommended medium volume with it, and plates can have their edge wells left empty.
- ④ Read the bench recipe: suspension and medium for each vessel, plus the totals. If a per-well volume comes out too small, a dilution is prescribed with it.
- ⑤ Check what comes next.
Cell Seeding Formula and practical notes
Seeding is one division: the cells you need divided by the density you have. What takes the work is either side of it — counting only the live cells, making a plate as one pot rather than well by well, leaving the edges out, and knowing what to do when the answer is a volume nobody can pipette.
The formula
Adherent and suspension differ only in the line that works out how many cells are needed.
- Live density (cells/mL)
- current density × viability ÷ 100
- Cells needed — adherent
- target (cells/cm²) × growth area (cm²)
- Cells needed — suspension
- target (cells/mL) × working volume (mL)
- Suspension (mL)
- cells needed ÷ live density
- Medium (mL)
- working volume − suspension
- Dilution
- live density ÷ density in the well
Leaving the edges out
Skipping the outer ring leaves (rows − 2) × (columns − 2) wells.
- 96-well (8 × 12)
- 6 × 10 = 60 wells — 36 left empty
- 48-well (6 × 8)
- 4 × 6 = 24 wells
- 24-well (4 × 6)
- 2 × 4 = 8 wells
- 12-well (3 × 4)
- 1 × 2 = 2 wells
- 6-well (2 × 3)
- 0 wells — every well is an edge well
Practical notes
- ⚠️ The confluency figures in the vessel table were measured with HeLa cells — the source says so in a footnote. They vary a great deal between cell lines, so do not carry them across unchanged.
- ⚠️ The 10% extra and the 2 µL pipetting floor are our defaults and are not quoted from any source. Do not cite them as sourced figures.
- Making one pot is not a convenience — it is a different answer. Filling wells individually means the error in each few-microlitre transfer lands directly on the cell count in that well.
- Edge wells evaporate faster, so they are usually left empty or filled with PBS. On a 96-well plate that leaves 60 usable wells.
- There are no cell line presets. No primary source for per-line seeding densities or doubling times could be obtained — ATCC's own product pages do not publish a doubling time — and these vary so much with conditions that a guessed default would be worse than none.
- Shake flasks and bioreactors are not listed either: no primary document for their recommended working volumes opened. Suspension work is not blocked by this, since the working volume is typed in directly.
- After seeding adherent cells, slide the plate back and forth rather than swirling it, or the cells collect in the middle.
FAQ
Choosing a vessel fills in what the source table recommends. That is a general figure, though — it depends on the cell line and on what you are doing. The biggest factor is when you need them: seed densely for tomorrow, sparsely for three days' time.
The answer itself. Filling wells one at a time often means a few microlitres of suspension per well, and the error in each of those small transfers lands directly on that well's cell count. Preparing one pot at the target density and dispensing equal volumes removes it. For plates, treat this as the default.
The outer ring loses medium to evaporation faster than the wells inside, so conditions drift apart across the plate. Leaving it empty, or filling it with PBS or sterile water, is common practice. On a 96-well plate that takes 36 wells out and leaves 60, and the calculation follows the 60.
Then it prescribes rather than just warning: how far to dilute the suspension and how many microlitres to take per well from that. The fold chosen is the smallest one that clears the floor, because every extra transfer step is another one to be wrong about. Raise the pipetting floor if you want more headroom.
No primary source for per-line seeding densities and doubling times could be obtained. ATCC's product pages do not publish doubling times, and the figures in expression-system guides are transfection-day densities that would be wrong as routine passage defaults. Given how much these vary with conditions, leaving them out beats shipping a guess as a default.
Dead cells are counted but do not grow, so the density is recalculated from the live ones. At 80% viability it takes 25% more suspension to deliver the same number of cells, and the page says so in a sentence.
References
Where the vessel table comes from. Surface area, medium volume, seeding density and cells at confluency were all taken from one document; the 60 values for 15 vessels were re-checked against it on 2026-08-21 and all matched.
- Gibco (Thermo Fisher Scientific). Useful Numbers for Cell Culture — Cell Culture Basics. Read 2026-08-21.https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.htmlThe whole table — four dishes, seven plates, four flasks. The footnote that the confluency figures are for HeLa cells is the document's own
Plate grids (6-well = 2 × 3 and so on) are not in that document; they are the SBS layouts. Working volumes for shake flasks and bioreactors are not in the table because no primary document for them could be opened.