Figure 2.

Generation of phenotypically mature mDC from adherent monocytes using the Pittsburgh Protocol in roller bottle cultures in comparison to static flask cultures. PBMC from a normal donor leukopheresis product was seeded into 850 cm² roller bottles or into T-175 flasks and the monocytes adhered for 2.5 h as described in the Methods section. After washing out the non-adherent cells in both systems, the cells were cultured for 4 days with 1,000 U/ml GM-CSF and 1,000 U/ml IL-4 and then matured using the Pittsburgh Protocol combination of cytokines. The floating cells were harvested after 24 h and stained for CD11c, CD14, HLA class II DP, DQ, DR, CD83, CD86, and CD80. The unstained and stained populations in the histograms are shown in grey and red, respectively. In the case of CD86 and CD83 staining, the surface expression on cells from non-matured cultures (in blue) is shown as a comparison to verify that maturation was induced in both systems. The results of one out of 3 similar experiments are shown.