Ovalbumin (OVA, Grade VII) was purchased from Sigma-Aldrich. For the analysis of the processing of liposome-coupled OVA by macrophages, DQ-OVA, which exhibits green fluores- Figure 2 . Uptake of liposome-coupled OVA by macrophages. Alexa-labeled OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages as described in Materials and Methods. Thirty minutes after the onset of the culture, macrophages were recovered and analyzed using flow cytometry. doi:10.1371/journal.pone.0015225.g002 Figure 1 . Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates. A, DQ-OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages expressing DM-DsRed (class II) or labeled with red fluorescein (class I), as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage DM or class I); cell only, macrophages without co-culture with DQ-OVA-coupled liposomes. B, the green-and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values 6 SD of the images shown in Fig. 1A . Asterisk, significant (p,0.01) difference of samples. doi:10.1371/journal.pone.0015225.g001 cence upon proteolytic degradation, was purchased from Molecular Probes, Inc. Synthetic CpG ODN (5002: TCCAT-GACGTTCTTGATGTT) was purchased from Invitrogen and was phosphorothioate-protected to avoid nuclease-dependent degradation.