Immature MoDCs store macropinocytosed Ag in late endocytic compartments. (A) MoDCs were pulsed for 1 hour with 10 μg/mL of fluorescent F(ab)′2 and washed and chased for 4 or 18 hours. After each time point, cells were fixed and amount of Ag was measured by flow cytometry. Results are expressed as a percentage of cells before chase. Data are mean ± SEM of 3 independent experiments. (B) MoDCs were treated as in panel A and then analyzed by wide field microscopy and deconvolution of Z-stack images. The percentage of colocalization of F(ab)′2 with CD63 or Lamp1-positive compartments was measured from deconvolved images of the whole cells. Data are mean ± SEM of 3 independent experiments (20-50 cells per condition). (C) MoDCs were treated as in panel B. A medial optical section is shown. Bar represents 5 μm. (D) Immature MoDCs were pulsed for 1 hour with 50 μg/mL F(ab)′2 and washed twice and chased for 4 hours before embedding for electron microscopy analysis. Immunogold labeling was performed to detect F(ab)′2 on thawed cryosections (10-nm gold particles). The white arrowheads indicate label in late endosomal vacuoles; and black arrowhead, label in a tubular structure. The black arrow points to a label in a tubular extension of an early endosomal vacuole. P indicates plasma membrane; E, early endosomal vacuole; G, Golgi; M, mitochondrium; and L, late endosomal/lysosomal vacuole. Bar represents 200 nm.
