Assessment of the surface charge of maturing phagosomes. (A–C) RAW macrophages were cotransfected with mRFP-Palm, a farnesylated and dually palmitoylated construct modeled after the tail of H-Ras and fused to mRFP and either GFP-8+ (A), GFP-5+ (B), or GFP-2+ (C). The cells were exposed to IgG-opsonized sRBCs. Confocal images were acquired 1 min, 5 min, or 1 h after initiation of phagocytosis. GFP fluorescence of the surface charge probes is shown in the main panels, whereas mRFP-Palm is shown in the insets. The asterisks denote the position of the sRBCs. Images in A–C are representative of at least 30 cells from two similar experiments. (D and E) Quantification of the fluorescence intensity of GFP-8+ and GFP-5+ (green bars in D and E, respectively) and mRFP-Palm (red bars) at the specified stage of maturation. Results are presented as the ratio of fluorescence intensity at the phagosome to that in the bulk, unengaged plasma membrane (PM). Data in D and E are means ± SD calculated from 10 cells from a representative experiment. Bars, 2 µm.