Immunofluorescence analysis on nonadherent cells generated in Lin⁻c-kit⁺ HPCs cultures. (A) Using a two-color immunofluorescence analysis, the proportion of Ia⁺DEC-205⁺ (▨) or Ia⁺CD86⁺ cells (▪) was determined on nonadherent cells generated from Lin⁻c-kit⁺ HPCs cultured with or without mouse TNFα combined with GM-CSF and SCF for 7 and 14 days, respectively. The data are expressed as the mean ± SD of four independent experiments or more. Error bars indicate 1 SD. *P < .05 significance as compared with the cultures without mouse TNFα addition. (B) Three-color immunofluorescence analysis on nonadherent cells generated in Lin⁻c-kit⁺ HPCs stimulated with mouse TNFα combined with GM-CSF and SCF for 14 days. These cells were sequentially stained with DEC-205 MoAb, biotinylated CD86 MoAb, and PE-labeled Ia MoAb. DEC-205 and CD86 were then shown by antirat IgG(Fab′ )₂-conjugated FITC and CY-streptavidin, respectively. The cell population that expressed high levels of Ia-antigen was gated and the coexpression of DEC-205 and CD86 was analyzed. More than 20,000 events were acquired. Three independent experiments were performed and representative results are shown here. The figures in each quadrant represent the percentage of the cells.