Fig. 7. Macropinocytosis-macropinosome discharge coupling. The two left histograms of panel A summarize FACS results with DCs loaded for 20 minutes with FITC-conjugated Bds and then split in two aliquots, one fixed immediately (left), the other after an additional 10 minutes of incubation with TRITC-conjugated Bds (centre). Notice the decrease of the FITC signal (over 30%) during the second incubation compensated by an analogous accumulation of the TRITC signal, indicating replacement of the discharged green Bds with red Bds. The histogram to the right of A shows that the 20 minutes of accumulation of FITC-conjugated Bds in DCs pretreated with Vac1 is almost 30% larger than that of non-pretreated cells, as expected for a drug-induced blockade of macropinosome discharge. The DCs of panels B and C, first pulsed for 20 minutes with FITC-conjugated Bds, were chased for 10 or 20 minutes with non-fluorescent Bds and then immunolabelled for EEA1 and TfR. Morphometric analyses of confocal images (at least ten fields) revealed considerable decreases of Bd fluorescence during chase (B), largely because of the discharge of Bd-rich puncta positive for EEA1 (C). By contrast, Bd-rich puncta positive for TfR alone increased proportionally (from 10% to 40% of the puncta present at 0 and 20 minutes of chase, respectively, panel C).