Phenotypic and cell proliferation analysis of monocytes cultured with GM-CSF plus IL-4 or M-CSF. (A) Histograms show the expression of the indicated markers by MHC II^int and MHC II^hi DCs from 3-day cultures of monocytes with GM-CSF plus IL-4 or M-CSF in the presence or absence of LPS (only the phenotype of MHC II^hi DCs is shown for cultures with GM-CSF plus IL-4 supplemented with LPS because MHC II^int DCs are almost absent in these cultures). The percentage of cells with a fluorescence intensity over the vertical lines (dark area of the gray profiles), corresponding to the upper limit of control background staining (white profiles shown in the CD86 histograms), is indicated. Data are representative of 4 experiments with similar results. (B) Cell proliferation in cultures of monocytes with GM-CSF and IL-4 was assessed after labeling with the intracellular fluorescent dye CFSE. Density plots show CFSE-versus-CD11c staining profiles of monocytes loaded with CFSE before culture, and the CFSE-versus-CD11c, -MHC II, and control background staining profiles of CFSE-loaded monocytes cultured with GM-CSF plus IL-4 for the indicated times. For each culture time, the total number of cells per well is indicated. CFSE dilution allowed us to assess whether cells had undergone 0, 1, or 2 cell divisions (1:1, 1:2, and 1:4 CFSE dilution, respectively). (C) Higher magnification of the CFSE-versus-CD11c profile from a 3-day culture showing DCs (D1) generated from monocytes that did not divide (M1), DCs (D2) generated from monocytes that had undergone 1 cell division (M2), and monocytes that had undergone 2 cell divisions (M3).