A limiting dilution assay to determine the frequency of antigen presenting cells

A limiting dilution assay to measure the functional frequency of antigen presenting cells (APC) has been developed and used to measure the frequency of mouse lymphoid cells which stimulated the mixed leucocyte response. The experiments utilised measurements of proliferation in 20 microliter hanging drop microcultures of activated T lymphocytes. When the data supported a model of single-hit kinetics, the APC frequency in the titrated stimulating cell population was calculated by the method of maximum likelihood. The assay was validated by measuring the frequency of unpurified spleen cells and enriched spleen dendritic cells which presented alloantigens in mixed leucocyte culture. APC frequency in unpurified spleen cells was 1:7254 (SE +/- 2134, 8 experiments) and was increased markedly to 1:15 (SE +/- 7, 7 experiments) when enriched populations consisting 70-95% of dendritic cells were used as stimulators.