Successive generation of immature and mature dendritic cells from monocytes. Monocytes (day 0) were cultured for 6 days in the presence of GM-CSF and IL-4 to induce Dc differentiation, followed by a three day culture period in the presence of GM-CSF, IL-4 and TNFα to induce DC maturation. Monocyte purity was determined by CD45/CD14 staining (day 0). The frequency of CD14negCD83neg immature DC, and CD14negCD83+ mature DC was determined after 6 and 9 day of culture by flow cytometry. Quadrants were set according to isotype controls.