The CD11b^−/dullCD11c⁺ and CD11b^+hiCD11c⁺ DC precursors display a different immunophenotype. Murine Lin⁻c-kit⁺ HPCs were cultured in presence of GM-CSF + SCF + TNF-α for 6 days. The phenotype of the CD11b^−/dullCD11c⁺ and CD11b^+hiCD11c⁺ precursors was determined by three-color immune staining using uncoupled test MoAbs shown by PE-conjugated anti-rat Ig and biotinylated anti-CD11c MoAb shown by CY-conjugated streptavidin, and the third color was shown by FITC-conjugated anti-CD11b MoAb. In some experiments FITC-conjugated anti-CD11b and PE-conjugated anti-CD11c MoAbs were used, while the test biotinylated MoAbs were shown by CY-conjugated streptavidin. In some experiments, uncoupled CD11b and biotinylated CD11c were shown by PE-conjugated anti-rat IgG and CY-conjugated streptavidin and finally FITC-conjugated test MoAbs. Histograms shown in the figures are gated on CD11b^−/dullCD11c⁺ and CD11b^+hiCD11c⁺ DC precursors. Solid and dotted lines indicate the immunofluoresecence intensity of cells stained with a control and the test antibodies, respectively. Representative results from three or more independent experiments are shown.