T-PCs are not hydrolytic compartments. (A and B) RAW cells ingesting FBTs cross-linked with DQ-ova. (A) Partially internalized FBT after 20 min of phagocytosis. Inset shows the lack of DQ-ova fluorescence. (B) Images from live-cell video microscopy showing fully internalized FBT undergoing degradation as detected by DQ-ova fluorescence (image acquisition using EM-CCD camera; Hamamatsu Photonics). External FBTs were labeled in the cold before imaging (not depicted). (C) Top: LysoSensor fails to label partially internalized FBTs (arrow), but accumulates around fully internalized FBTs (bottom panels). 20 min after phagocytosis external FBTs were immunolabeled in the cold (pink). Lysosensor was added (2 min) and cells moved to precooled microscope stage. White lines indicate cell boundaries. (D) The number of partially and fully internalized FBTs positive for lysosensor from C. Data shown are means ± SEM from three independent experiments; n = 25. (E) V-ATPase proton pumps surround the T-PCs. Phagocytosis of FBTs by cells expressing voa3-GFP (white) subunit of V-ATPase. Cells were fixed after 20 min of phagocytosis and external FBTs were labeled (pink, inset). Bottom panels: magnified single planes from framed regions. (F) Cathepsin-D in cells internalizing FBTs. Cells were fixed at indicated times, F-actin was stained (green), and cathepsin-D was immunolabeled (white). The percentage of FBTs positive for cathepsin-D at each time point is indicated. Data shown are means from two independent experiments. Atleast 25 FBTs were assessed in each case. Bars: (main panels) 5 µm; (magnifications) 2.5 µm.