Cell Seeding Density Calculator
How many cells to seed per well, dish or flask — by cells/cm², cells per vessel or % confluence — with the stock and medium volumes for a master mix or per-well pipetting, viability correction, a scale-up table for every plate format and a projected confluence curve.
How it works
Cells per vessel = density (cells/cm²) × growth area · density from % confluence = % × confluent density · total cells = cells per vessel × number × (1 + extra%) · stock volume = total cells ÷ (stock concentration × viability) · master mix = medium per vessel × number × (1 + extra%) · time to confluence = lag + doubling time × log₂(confluent density ÷ seeding density)
Seeding by cells per cm² keeps confluence timing the same in every format: a 6-well well has about 30 times the area of a 96-well well, so it needs 30 times the cells. You can start from a density, from a protocol’s “cells per well”, or from the confluence you want right after attachment (using the cell line’s density at 100% confluence — about 1.2 × 10⁵ cells/cm² for HeLa). The calculator then works out how much counted stock to take, adding a margin for pipetting loss, and either a master mix you split between the wells or the stock to pipette into each well with the medium to top it up. If your count includes dead cells, enter the viability so the target is met with live cells. The scale-up table shows the same density in every common plate, dish and flask, and the growth projection uses the doubling time to estimate when the culture will reach confluence.
Worked example
Six wells of a 6-well plate at 20,000 cells/cm²: 20,000 × 9.5 cm² = 1.90 × 10⁵ cells per well; with 10% extra that is 1.90 × 10⁵ × 6 × 1.1 = 1.254 × 10⁶ cells. From a stock of 1.2 × 10⁶ cells/mL take 1.045 mL and add 18.76 mL of medium for 19.80 mL of mix, then plate 3 mL per well. Seeding sits at 17% of a 1.2 × 10⁵ cells/cm² confluent layer, and with a 24 h doubling time the wells reach confluence after about 2.6 days.
Sources: Thermo Fisher Gibco — Useful numbers for cell culture (vessel areas, seeding and confluent cell numbers) · NEB — How many cells should I seed into cell culture plates?
Frequently asked questions
Cells per well vs cells per cm²?
Papers often quote cells per well for one plate type. Choose “Cells per vessel” and the plate they used to get the density, then switch the vessel to see the matching number for your format — or read it from the scale-up table.
How much medium per well?
The defaults are typical working volumes (0.2 mL in a 96-well plate, 3 mL in a 6-well plate, 12 mL in a T75). Enter your own volume under More options if your protocol differs; the master mix and the cell concentration in the wells update.
Master mix or pipetting stock into each well?
A master mix (cells diluted into the full volume, then split) gives the most even wells and is the better choice when the stock volume per well is small. Pipetting stock straight into wells that already hold medium is quicker for a few flasks; mix by rocking, not swirling, so cells do not pile up in the centre.
How many cells go in a T75 flask?
At 20,000 cells/cm² a T75 (75 cm²) takes 1.5 × 10⁶ cells. Many lines are split to about 2–3 × 10⁴ cells/cm² and reach confluence in 3–4 days; check the supplier’s recommendation for your line.
How is time to confluence estimated?
From the seeding density, the density at 100% confluence and the doubling time: each doubling time the cell number doubles, so the time is lag + doubling time × log₂(confluent ÷ seeding density). It is an estimate — growth slows near confluence and after a harsh passage.
Related calculators
For teaching and planning — verify against your lab protocol and instrument documentation.