Figure 7.

Phagocytosis of neuronal fragments by differently activated macrophages. Phagocytosis of fluorescently labeled neuronal fragments by activated bone marrow derived macrophages was determined using flow cytometry. Phagocytosis was blocked using anti-MAC-1 antibodies, luteolin, quercetin and DPI. Data are the mean of 4 separate experiments (n = 4) ± SEM. *= p < 0.05 and #= p < 0.05. A) CA macrophages phagocytosed significantly more neuronal fragments compared to control and AA macrophages. B-D) Visualization of neuronal fragment internalization by phagocytosis, red: DiI labeled neuronal fragments; green: Mac-1 on macrophage membrane. Representative images, taken on a confocal microscope (Leica TCS SP2), are shown. Red labelled neuronal fragments are clearly present within the green labelled membrane of the differently activated macrophages. Some binding is also present, with the neuronal fragments being present outside the membrane. A 20 times magnification was used. E) To determine whether phagocytosis could be blocked macrophages were exposed to luteolin, quercetin, MAC-1 (anti-CR3 antibody) or DPI (NADPH oxidase blocker). Phagocytosis of neuronal fragments by all types of macrophages was significantly reduced by exposure to luteolin and quercetin. Using MAC-1 antibody did not reduce neuronal fragment phagocytosis. DPI led to a significant reduction in control macrophages, however, CA and AA macrophages did not reduce phagocytosis significantly.