Acidification of macrophage phagosomes parallels recruitment of the V-ATPase to the phagosomal membrane. (A) RAW264.7 cells were incubated with IgG-opsonized FITC-zymosan to initiate phagocytosis. Phagosomal pH changes were determined by ratiometric fluorescence microscopy. Time 0 marks the point where acidification became evident. Data points (open circles) represent the average of two independent experiments, with ≥10 cells per replicate. (B) Schematic showing the stages of phagosome engulfment and maturation, with associated membrane markers. (C) V-ATPase localization in resting RAW264.7 cells stained with SidK-AL568 (magenta). (D–H) RAW264.7 cells transfected with the indicated phagosomal maturation markers (green) were allowed to internalize IgG-opsonized SRBCs for various times before fixation. Cells were then fixed, permeabilized, and stained with SidK-AL568 (magenta). (D) Cells expressing a PM marker, fixed 2 min after initiation of phagocytosis. (E) Cells expressing Rab5A, fixed 5 min after initiation of phagocytosis. (F) Cells expressing a PtdIns(3)P-specific probe, fixed 5 min after initiation of phagocytosis. (G) Cells expressing a Rab7 marker, fixed 30 min after initiation of phagocytosis. (H) Cells immunostained for LAMP1, fixed 30 min after initiation of phagocytosis. (C–H)XY optical slices acquired near the middle of the cell or phagosome, representative of ≥30 fields from three or more experiments of each type. Scale bars: 5 µm.