Inhibition of phagocytosis in HIV-1–infected human macrophages. (A) Primary human macrophages were infected with HIV-1_ADAWT or HIV-1_ADAΔNef or were noninfected for 8 days. The cells were incubated for 60 minutes with IgG-RBCs at 37°C, then fixed and stained with Cy2-anti–rabbit IgG to reveal external RBCs (iv-vi). Particles internalized in closed phagosomes are also detected by phase contrast (vii-ix). The cells were then permeabilized and labeled with anti-p24, followed by Cy3-anti–mouse IgG. Merged images show p24 in green and RBCs in red (i-iii). Arrowheads indicate RBCs in phagocytic cups. Z stacks of wide-field fluorescent images were acquired using a piezo, and 1 medial optical section is shown. The stacks of images are presented as 3-dimensional reconstructions (right panel). Bar represents 10 μm. (B-C) HIV-1_ADAWT or HIV-1_ADAΔNef–infected cells were incubated for 60 minutes at 37°C with IgG-RBCs, C3bi-RBCs, or Cy3-zymosans. The cells were then treated as in panel A except that they were labeled with anti-p24, followed by Cy2-anti–mouse IgG when Cy3-zymosan was used. The efficiencies of association (B) and phagocytosis (C) were calculated for 50 infected and 50 noninfected cells (control). Results are expressed as a percentage of control cells. The means ± SEM of 3 independent experiments are plotted. (D-E) Primary human macrophages transiently expressing GFP or Nef-GFP were incubated with IgG-RBCs or C3bi-RBCs for 60 minutes at 37°C. The cells were then fixed and stained as in panel A. The efficiencies of association (D) and phagocytosis (E) were calculated and expressed as in panels B-C. The means ± SEM of 3 independent experiments are plotted.