LimitingDilution

Limiting-dilution, also called limiting-dilution assay (LDA), is a quantitative method used to estimate the frequency of cells within a mixed population that have a specific functional capability, such as colony formation, cytokine production, or stem-cell activity. The core idea is to dilute the cell suspension across many wells or vessels so that each well contains zero, one, or more target cells, enabling statistical inference of the prevalence of the activity.

The method relies on Poisson statistics: if the average number of target cells per well is λ, the probability that a well is negative (no functional cell present) is e^{-λ}. By plating at several dilutions and recording the fraction of negative wells at each dilution, the frequency f of functional cells can be estimated, typically via maximum-likelihood fitting.

Typical procedure involves preparing a single-cell suspension with viability checked; performing serial dilutions; plating multiple wells at each dilution with appropriate cell numbers; incubating under conditions that reveal the readout (for example, colony growth, secretion, or proliferation); and scoring wells as positive or negative. Data are then used to compute frequency.

Readouts include colony-forming unit assays, limiting-dilution cloning, or immunoassays (such as ELISPOT variants) that indicate activity in a well. Limitations include the need for random distribution, consistent viability, and sufficient sample size; clumping or differential survival can bias results.

LDA is used in hematology, immunology, and cancer research to quantify stem or progenitor cell frequencies and to isolate clones by extreme dilution.