FIGURE 6. Confocal microscopy and SM4s enhancement of uptake of apoptotic cells by macrophages.

A, DiO-labeled apoptotic cells were cocultured with macrophages at 37°C for 1 h. The macrophages were washed from noningested apoptotic cells, stained with Alexa-546 phalloidin (actin) and DRAQ5 (nucleus), and visualized with confocal microscopy. Data are presented in red and blue channel (Alexa-546 and DRAQ5, respectively; left), green channel (DiO; middle), and merge (right). B, Higher magnification confocal images of macrophages with intracellular localization of apoptotic material (×2500). C, Percent of phagocytosis-active macrophages was calculated by dividing numbers of DiO-positive and negative macrophages, multiplied by 100. D, Single macrophages were analyzed for the intensity of green fluorescence, as a parameter of intensity of phagocytosis. Mean green fluorescence intensity of macrophages from macrophages + C26 samples was taken as “standard” (100%). Fluorescence intensity of single macrophages was calculated as percentage of standard 20 macrophages per condition per experiment were analyzed. All data are presented as mean ± SEM of three experiments in duplicates. Statistical differences were evaluated by Mann-Whitney U test. *, p < 0.05 and ***, p < 0.001.