How do you design a dose-response experiment?
Start from the question: ranking compounds, confirming activity, or measuring a potency you will report. That decides how many concentrations you need and where they sit. For a reliable relative IC50 or EC50, Sebaugh (2011) recommends at least two concentrations on each plateau, beyond the lower and upper bends of the curve. For an absolute IC50, at least two concentrations should give responses below 50% and two above 50%.
So the concentration range has to bracket the expected potency on both sides, with enough points to define both plateaus. Spacing is usually constant on a log scale, for example half-log or threefold steps. The Serial Dilution Calculator works out the series and the transfer volumes.
What controls does a cell-based assay need?
Every plate should carry the controls that define its window: a negative or vehicle control at the same vehicle concentration as the test wells, a positive control or reference compound, and background wells where the readout needs them. Controls on every plate let each plate be normalized on its own, which matters when plates are read on different days.
For screens, the Z′-factor described by Zhang and colleagues (1999) summarizes how well the positive and negative controls separate, given their variability. A Z′ of 0.5 or above is conventionally taken as an excellent assay; between 0 and 0.5 is marginal; below 0 the controls overlap too much to screen. Agree the acceptance criteria before the first plate is run.
How many test wells will the assay use?
The planner multiplies cell lines x compounds x concentrations x replicates for each time point, then by the number of time points you list. It does not add control, blank or edge wells, because those depend on your plate layout. To lay the wells out on 96- or 384-well plates, use the Plate Layout Planner, and the Cell Seeding Calculator to work out the cell suspension.
Which checks should come before the first plate?
Cell line identity is the classic failure. The ICLAC Register lists hundreds of misidentified or cross-contaminated cell lines, and authenticating human lines by STR profiling is the accepted check. Add mycoplasma testing, the maximum vehicle concentration the cells tolerate, compound solubility at the top concentration, and the curve-fitting method and software. The NCATS Assay Guidance Manual is a free, detailed reference on assay development and validation.
Frequently asked questions
How many concentrations do I need for an IC50?
Enough to define both plateaus of the curve. Sebaugh (2011) recommends at least two concentrations beyond each bend of the curve for a relative IC50, and at least two responses below and two above 50% for an absolute IC50. Many labs run 8 to 12 points, but the right number depends on how wide the range must be.
What is the Z′-factor and how is it calculated?
Z′ = 1 - 3 x (SD of positive control + SD of negative control) / |mean of positive control - mean of negative control|. It measures assay window against noise. Zhang et al. (1999) describe 0.5 or above as excellent and below 0 as unusable for screening.
How many replicates should an in vitro assay have?
Technical replicates (wells) estimate plate noise; biological replicates (independent experiments or passages) show whether the result repeats. Decide both with your team based on the decision the data will support. The planner records your choice and counts the wells.
What is the difference between in vitro and in vivo studies?
In vitro studies use cells, tissues or biochemical systems outside a living organism; in vivo studies use living animals. In vitro work is faster and cheaper per data point and is used to screen and rank; in vivo work tests efficacy, exposure and safety in a whole organism.
Why authenticate cell lines?
Because misidentified and cross-contaminated lines are common enough to have a public register (ICLAC). STR profiling confirms a human cell line's identity. Without it, results may describe a different cell line from the one named.
Is anything I type stored or shared?
No. The planner runs entirely in your browser. Nothing you enter is saved, sent to BioBridgeX, put in the page address or passed to analytics or AI services. Closing or refreshing the page clears it, so copy or print the plan before you leave.
Is the generated plan a protocol?
No. It organizes the requirements you entered into a structured outline and lists what is still undecided. It is not a validated protocol, it does not recommend sample sizes, and it does not establish GLP, GMP or regulatory compliance. Have the plan reviewed by qualified scientific, statistical, ethical and regulatory experts.
Related free tools
- Organoid Drug Screening Planner: Tumor type, organoid models, compounds, dose range, controls, exposure and readouts as a screening plan.
- Serial Dilution Calculator: Transfer and diluent volumes and a concentration table for 2-fold, 3-fold, half-log or 10-fold series.
- 96 and 384 Well Plate Layout Planner: Lay out compounds, concentrations, replicates and controls on 96- or 384-well plates and count the plates.
- Cell Seeding Density Calculator: Cells per well or per cm² into the cell suspension and medium to mix for a plate, with spare volume.
- All study planners and every free tool on BioBridgeX.
Sources
- Sebaugh JL. Guidelines for accurate EC50/IC50 estimation. Pharmaceutical Statistics 2011;10(2):128-134
- Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4(2):67-73
- Markossian S et al., eds. Assay Guidance Manual. Eli Lilly & Company and NCATS, 2004-
- ICLAC Register of Misidentified Cell Lines
This planner organizes requirements. It is not a validated protocol, does not recommend sample sizes, and does not establish GLP, GMP or regulatory compliance. Review every plan with qualified scientific, statistical, ethical and regulatory experts. Facts on this page last checked 27 October 2026.
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