To investigate the localization of OVA-liposome conjugates by macrophages, macrophage clone 39 or DM-DsRed-expressing cloned macrophage 39 was cultured for 18 h at 37uC on 8-hole heavy Teflon-coated slides (Bokusui Brown) and was then incubated with DQ-OVA-liposome conjugates, prepared using oleoyl or stearoyl liposomes, for 2 h at 37uC. The slides were then washed with MEM and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, they were incubated for 10 min in 0.1 M glycine-HCl (pH 7.0) to block the remaining aldehyde residue. They were then washed two times in PBS. After washing, the slides were sealed with PBS:glycerin (1:9) and analyzed under an LSM510 confocal laser scanning microscope system (Zeiss). For analysis of co-localization of OVA and MHC class I, early endosomal antigen 1 (EEA1) or lysosomalassociated membrane protein-1 (LAMP-1) after blocking of the remaining aldehyde residue, cloned macrophage 39 was subsequently permeabilized with 0.05% saponin-TBS for 10 min at room temperature. After being washed twice with PBS, they were reacted with biotin-conjugated mouse anti-mouse H-2D d mAb (34-2-12, 10 mg/ml; BD Biosciences), goat anti-mouse EEA1 Figure 5 . IFN-c production by splenic CD4/CD8 + T cells of mice immunized with OVA after co-culture with CD11c + cells pulsed with OVA coupled to oleoyl liposomes. Splenic CD4/CD8 + T cells were taken from mice immunized with OVA and were cultured with CD11c + cells pulsed with OVA coupled to oleoyl liposomes with or without inhibitors as described in Materials and Methods. IFN-c production of T cells in the supernatants in the absence of inhibitors was normalized to 100%. Data represent the mean values 6 SD of triplicate culture. Asterisk, significant (p,0.01) difference as compared with the 'no inhibitor' group. doi:10.1371/journal.pone.0015225.g005 polyclonal antibody (N19, 1 mg/ml; Santa Cruz Biotechnology) or rat anti-mouse LAMP-1 monoclonal antibody (1D4B, 1 mg/ml; Santa Cruz Biotechnology) for 18 h at 4uC. After being washed three times with TBS, they were reacted with Alexa 546conjugated streptavidin (1:200 diluted; Invitorogen) to detect MHC class I, Alexa Fluor 568-labeled Ab (rabbit anti-goat IgG, 10 mg/ml; Invitrogen) to detect EEA1 or Alexa Fluor 568-labeled Ab (goat anti-rat IgG, 10 mg/ml; Invitrogen) to detect LAMP-1 for 4 h at room temperature. They were then washed two times in TBS. After the washing, the slides were sealed with PBS:glycerin (1:9) and analyzed under an LSM510 confocal laser scanning microscope system (Zeiss).