V-ATPase accumulates in subdomains along the phagosomal membrane and is excluded from ER contact sites. (A) RAW264.7 cells transfected with ORP1L-GFP were allowed to internalize IgG-opsonized SRBCs and fixed after 60 min. Cells were stained with SidK-AL568 (magenta) and visualized along with ORP1L (cyan). Individual SidK-AL568 and ORP1L channels and the SidK-AL568:ORP1L fluorescence ratio (Ratio_{SidK-AL568:ORP1L}) are shown. Fluorescence ratio is pseudocolored in a rainbow look up table, corresponding to ratio values from 0 to 2. (B) RAW264.7 cells transfected with Arl8b-GFP allowed to internalize IgG-opsonized SRBCs, fixed after 30 min, then stained with SidK-AL568 (magenta) and visualized along with Arl8b-GFP (cyan). Individual channels and the pseudocolored fluorescence ratio are shown. (C) Human macrophages were allowed to internalize IgG-opsonized SRBCs and fixed after 30 min. Cells were fixed, permeabilized, and stained with SidK-AL568 (magenta) and immunostained for endogenous VapB (cyan). Smaller panels show (top to bottom) individual SidK-AL568, VapB, and SidK-AL568:VapB fluorescence ratio channels (Ratio_{SidK-AL568:VapB}) for three individual phagosomes (left to right) identified by dotted squares, all at 1.8× magnification. (A–C) Central 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.