A serial dilution calculator for cell-based assays.
This serial dilution calculator builds the transfer and diluent volumes for a dose-response series and holds the solvent fraction identical in every well, so concentration is the only thing that changes across the plate. It returns a pipettable two-stage scheme, working-solution volumes with overage, a plate map and a bench protocol.
Bar height is the solvent each well receives. Top: vehicle tracks concentration, so the dose-response carries two variables. Bottom: the series is built in neat solvent, then a fixed volume goes into a fixed volume of medium.
What is a serial dilution calculator?
A serial dilution calculator works out the transfer and diluent volumes needed to step a stock solution down through a fixed ratio, with each tube made from the one before it. This one adds the constraint that decides whether a cytotoxicity plate is interpretable: the solvent fraction is held identical in every well, so concentration is the only variable across the series.
Cn = C0 ÷ f^n · In = Cn × Vwork ÷ vspike · vehicle % = 100 × vspike ÷ Vwork
Plan the series and the plate together.
Enter a stock, a concentration range and a plate format. The serial dilution calculator returns a scheme you can execute without a sub-microlitre transfer anywhere in it, and tells you when no scheme can work at all.
Experiment setup
Step by stepPlant and herbal extracts have no single molecular weight, so keep those in µg/mL.
Nobody makes 820 µL of a treatment. A 2 mL floor is what lets a 10 µL spike land at 0.5 % vehicle, so batch size and solvent ceiling are the same decision.
Every volume, in the order you pipette them.
Stage one is prepared in neat solvent and never touches cells. Stage two is prepared in medium immediately before dosing. Rows shaded amber depart from the standard scheme because a transfer would otherwise have fallen below the pipetting floor.
Where each condition sits.
Conditions run across the columns and replicates run down the rows, which is how a plate is laid out in practice and how a multichannel reads it. Hover any well to see its contents.
How the calculation runs.
Every dilution here reduces to conservation of solute, written C1V1 = C2V2. What the serial dilution calculator adds is the constraint set that turns an arithmetically correct answer into one you can actually pipette.
Concentration of each point
For a constant fold series with factor f, the concentration at step n counting from zero is a geometric step. For a log-spaced range across k points, the ratio between neighbours is fixed rather than the arithmetic gap. The naming follows the usual convention for logarithmic and half-logarithmic dilution, where a ten-fold step is a log dilution and the 3.162-fold step offered above is a half-log.
f = (Ctop ÷ Cbottom)^(1/(k−1))
Working backwards from the well
The calculation starts at the well and works outwards, which is the reverse of how most calculators run. A fixed spike volume enters a fixed working volume, so the vehicle fraction becomes a single number for the whole plate.
In = Cn × Vwork ÷ vspike
vehicle % = 100 × vspike ÷ Vwork
The solvent floor
Substituting the requirement that the top intermediate cannot be stronger than the stock gives a bound that no arrangement of volumes can beat. When this floor sits above the tolerated ceiling, the design is impossible and the calculator says so rather than returning a number.
What the calculator refuses to return
These are the failures that show up in real plates, and each one blocks a clean answer.
Solvent tolerance and assay design
The vehicle ceiling used by default reflects the range at which dimethyl sulfoxide is generally reported to leave adherent line viability unchanged over a 24 to 72 hour exposure. Confirm the tolerated concentration for the specific line, passage and exposure duration, and run a vehicle control at the working concentration on every plate.
- International Organization for Standardization. (2009). ISO 10993-5: Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity (3rd ed.). ISO.
- Clinical and Laboratory Standards Institute. (2018). M07: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically (11th ed.). CLSI.
Serial dilution calculator questions and answers.
Direct answers on solvent limits, pipetting floors, curve geometry and the designs this calculator will not return.
What is a serial dilution calculator?+
A serial dilution calculator works out the transfer and diluent volumes needed to step a stock solution down through a fixed ratio, with each tube made from the one before it. This one adds the constraint that matters in cell work: the solvent fraction is held identical in every well, so concentration is the only variable across the series.
How do you keep the DMSO the same in every well?+
Build the whole series in neat solvent first, then add the same fixed volume of each intermediate to the same volume of medium. If 10 µL of every intermediate goes into 1.99 mL of medium, making 2.00 mL final, every well carries 0.5 % v/v vehicle. Diluting the stock straight into medium at a different ratio for each point does the opposite: in an eight-point two-fold series the top well ends up with 128 times the solvent of the bottom well.
What is the maximum DMSO concentration for cell culture?+
Most adherent lines tolerate 0.1 to 0.5 % v/v over 24 to 72 hours without a measurable drop in viability, and 0.5 % is the usual working ceiling for cytotoxicity work. At 1 % the solvent itself starts contributing to the response. Primary cells and suspension lines are less forgiving, often 0.1 %. Whatever limit you use, a vehicle control at that exact concentration belongs on the same plate.
What is the smallest volume I should pipette?+
Five microlitres is a practical floor for an air-displacement pipette. Below that, tip wetting, residual volume and surface tension produce a relative error large enough to matter, and an error at the top of a serial chain propagates into every point below it. If a scheme calls for less than 5 µL, add an intermediate step instead.
How many concentrations does an IC50 need?+
A four-parameter logistic fit has four unknowns, so five points is the arithmetic minimum and seven or eight is normal practice. The series has to straddle the IC50 with points on both plateaus, which usually means spanning three to four log units. A curve fitted only to the descending limb returns an interval wider than the range tested.
Why does the calculator refuse some combinations?+
The solvent carried into a well can never fall below 100 × top concentration ÷ stock concentration, whatever volumes are chosen. When that floor sits above the tolerated ceiling, no arrangement of volumes rescues the design, and the only fix is a more concentrated stock or a lower top dose. The calculator reports the stock strength that would work.
Can I use this for antibacterial MIC work?+
Yes for planning the series, with one difference in execution. Broth microdilution is done in-plate: 100 µL of broth in every well, 100 µL transferred along the row and 100 µL discarded from the last well, so the transfer volume equals the well volume. Set the well volume and spike accordingly and read the tables as tube volumes.
Does anything leave my browser?+
No. The calculation is JavaScript running in this page. Nothing is uploaded, stored or logged, which is why it keeps working offline once the page has loaded.
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