Calculation walkthrough
What a 5 µL seeding plan actually means
Compare 384- and 1536-well preparations, calculate a batch allowance and record which volumes your equipment actually dispenses.
Scientific contributor: Dr. Tayner Rodriguez Garzon · Published by Cell Culture Tools · How content is prepared
Interactive bench example
Which volume will your equipment dispense?
Prepared by Cell Culture Tools with AI-assisted drafting. Presets demonstrate arithmetic, not results observed in Tayner’s laboratory. Change the inputs to explore your plan, then record your own observations below.
Start with a 1536-well or 384-well demonstration. The 10% allowance is an example, not an equipment specification. Change it to match a documented requirement; the added cell and medium volumes must keep the same ratio.
- Stock contribution per well
- 1.5 µL
- Medium contribution per well
- 3.5 µL
- Prepare stock
- 2.5344 mL
- Prepare fresh medium
- 5.9136 mL
- Total with allowance
- 8.448 mL
- Viable cells needed with allowance
- 2,534,400
Bulk mixture: Combine 2.5344 mL stock with 5.9136 mL medium, then dispense 5 µL of the uniform mixture per well. The 1.5 µL stock contribution is not a separate per-well dispense.
The mistake: adding allowance as medium alone
In the 1536-well preset, the base mixture contains 2.304 mL stock and 5.376 mL medium. Adding 0.768 mL medium alone makes 8.448 mL but dilutes the dose to about 1,364 viable cells per 5 µL, instead of 1,500. Scaling both components gives 2.5344 mL stock plus 5.9136 mL medium and preserves the target.
The example plans quantities. It does not measure suspension uniformity or delivered cells. Record the actual instrument and any dispensing or count checks separately; a balanced batch is not an observed plating result.
Record what happened at your bench
Keep the calculation separate from the observation. These fields start blank. Entries stay on this page and are not submitted or saved by the app; download them before leaving.
Evidence to keep with this calculation
- Record the exact plate product, liquid handler or pipette, and the volumes actually dispensed.
- Document how the allowance was chosen, including reservoir and transfer requirements.
- Record the sampling positions and timing of any measured cell-distribution checks; do not infer uniformity from the calculation.
The download includes the current inputs, outputs, notes and an uncompleted review field. Entering a note does not certify the result or publish it on this website.
The full calculator does not add this worksheet’s allowance automatically. This worksheet checks the extra volume and cells explicitly.
One well, two volume descriptions
A final well volume of 5 µL does not specify the cell-suspension contribution. That contribution also depends on the viable cells wanted in the well and the viable concentration of the stock.
For an illustrative target of 1,500 viable cells/well and a stock of 1,000,000 viable cells/mL, the stock contribution is 1,500 ÷ 1,000,000 = 0.0015 mL, or 1.5 µL. Fresh medium supplies the remaining 3.5 µL. Their sum is the 5 µL final volume.
The same 5 µL target can give very different stock volumes
Hold the target at 1,500 viable cells/well and change only the viable stock concentration:
| Viable stock, cells/mL | Stock, µL | Fresh medium, µL | Interpretation |
|---|---|---|---|
| 2,000,000 | 0.75 | 4.25 | Below the app’s 1 µL stock threshold |
| 1,000,000 | 1.5 | 3.5 | No below-1-µL stock reminder |
| 800,000 | 1.875 | 3.125 | More stock is needed for the same cell target |
| 250,000 | 6 | Impossible | The stock alone exceeds the 5 µL final volume |
The last row is a feasibility failure, not a rounding problem. A workable plan would need a different measured stock concentration, cell target or protocol-approved final volume. The calculator cannot choose those biological conditions for you.
Apply viability once
If a counter reports 1,000,000 total cells/mL at 80% viability, the viable stock is 800,000 cells/mL. This gives the third row above. If it already reports 800,000 live cells/mL, enter that value with 100% in the calculator’s viability field. Applying 80% again would incorrectly reduce the stock to 640,000 viable cells/mL.
Check whether the reported concentration already accounts for the counting-sample dilution. A counter’s total/live labels and dilution settings are inputs to this decision; the website cannot infer them from the number alone.
Separate additions or a bulk mixture?
With separate additions, 1.5 µL stock and 3.5 µL medium are two individual dispensing steps. With a uniform bulk mixture, they describe the contributions to one 5 µL aliquot. The bulk method does not require a separate 1.5 µL dispense into every well.
For 1,536 wells at the first example’s concentrations, the mathematical batch is 2.304 mL stock + 5.376 mL medium = 7.680 mL. It contains 2,304,000 viable cells. Each uniform 5 µL aliquot represents 1,500 viable cells.
The below-1-µL reminder evaluates the stock contribution, not the final dispense and not your equipment’s accuracy. Use the plan to identify the actual volumes your equipment must deliver. Uniformity and delivered cell counts remain experimental questions; arithmetic alone does not verify them.
Extra batch volume must preserve the ratio
The seeding calculator does not automatically add dead volume or overage. As a numerical example only, a 10% allowance multiplies both components by 1.10: 2.5344 mL stock + 5.9136 mL medium = 8.448 mL. The final cell concentration stays at 300,000 viable cells/mL, and the prepared batch requires 2,534,400 viable cells.
Adding only extra medium would dilute the cells. Choose the allowance from your actual workflow; 10% is not a recommendation. To represent at least 10% extra as whole 5 µL dispensing units in the calculator, round 1,536 × 1.10 up to 1,690 units. That plans 8.450 mL, slightly more than the exact 10% example.
Open Seed cells →To reproduce the base case: 1536-well format; 1,536 wells; 5 µL final/well; cells/well target 1,500; stock 1,000,000 cells/mL; viability 100%; available stock 3 mL. The link opens the calculator; enter these illustrative values yourself.
Plate references and source scope
The Corning vessel guide, page 2, lists 25–50 µL for its standard 384-well format and 5–10 µL for its clear flat-bottom 1536-well format. The guide also distinguishes low-volume 384-well plates. Those product-specific ranges explain the app’s presets; they are not specifications for every plate.
The Countess II FL quick reference provides an example of total/live concentration reporting and dilution settings. Consult your own instrument’s instructions. Sources checked 19 October 2026. All numerical comparisons on this page are original arithmetic examples.