Generation of stable, mature DCs using different maturation cocktails. (A) Percentages of DCs harvested after primary cell culture (6 d differentiation + 24 h maturation) calculated on total seeded cells (mononuclear cells) or CD14-positive monocytes detected by FACS and manual counting. DC populations DC1–DC5 were matured in different cocktails as listed in Table 1. Viability was detected by 7AAD exclusion measured by flow cytometry in FL-3 of the FACS-Calibur and viable cells are expressed as percentages of total cells. Percentages of cells expressing various surface markers were determined by flow cytometry using the antibodies specified in "Methods", including CD14 (a monocyte marker), CD83 (a marker of mDCs), CD80 and CD86 (costimulatory molecules) and chemokine receptor 7 (CCR7 = CD197) as an indicator for DC migratory potential into lymph nodes. Data are expressed as percentages of total cells with acquisition of 1 × 104 events. CCR7 values presented here (Fig. 2A) represent the percentages in histograms overlayed by IgG2a isotype control, although generated from the same FACS stain they are slightly different to the CCR7 values shown as dot plots in Figure 2B. Broken lines indicate marker levels for DCs matured with DC1 (Jonuleit) cocktail. (B) Representative dot plots of DC1–DC5 populations showing percentages of cells positive for CD83 versus CCR7 and CD80 versus CD86. (C) DCs were washed free of maturation cytokines and cultured an additional 44 h in culture medium without cytokines. Viability was determined by 7AAD incorporation. Percentages of CD14, CD83, CD80, CD86 and CCR7 were determined as described above. Broken lines indicate marker levels for DCs matured with DC1 (Jonuleit) cocktail.