Effect of MARCO expression in antigen internalization. (A) D1 cells infected with the MARCO-expressing (MARCO) or the empty PINCO vector (control) were incubated with Ds-red E coli for 1.5 hours. Random fields of cells were counted and scored for the number of bacteria internalized (top panel) or bound to the cell membrane (bottom panel) by confocal microscopy. For each cell, horizontal and vertical sections were analyzed to count only internalized bacteria. The experiment was repeated twice with similar results. Error bars represent the standard deviations from the mean calculated on the indicated number of cells. (B) D1 cells infected with the MARCO-expressing (MARCO) or empty PINCO vector (control) were incubated for 20 minutes with PE-conjugated microbeads with a cell-bead ratio of 1:100. Uptake of microbeads at 4°Cor37°C has been measured on gated green cells by flow cytometry. MARCO-expressing cells show a reduced phagocytic activity. (C) GM-CSF– or M-CSF–grown microglial cells were treated as in panel B, and the efficiency of the beads' uptake at 4°Corat37°C was investigated by flow cytometry. GM-CSF–treated microglial cells show a reduced phagocytic activity. The experiment was repeated 3 times with similar results. Percentages in panels B and C represent the percentage of cells that have internalized beads.