Figure 3

MFG-E8 rescues macrophages from acute alcohol exposure—induced impairment of efferocytosis. Peritoneal macrophages were divided randomly into groups as indicated in the figure. They were subjected to treatment with culture medium alone (control) or medium containing alcohol (25 mmol/L) for 18 h respectively. After treatments, macrophages were further cocultured with apoptotic thymocytes in the 1:4 ratio for 90 min. In the group MFG-E8 + alcohol, the coculture medium contained 0.5 µg/mL of MFG-E8. At the end of treatments, all macrophages were processed for appropriate staining followed by determining macrophages that engulfed apoptotic cells using either a fluorescent microscopy or flow cytometry—based method as described in the Figure 2 legend. (A) Representative photomicrographs of TUNEL-stained samples visualized under a fluorescent microscope. Nuclei of apoptotic cells engulfed by macrophages were demonstrated with bright green fluorescence. Original magnification: 10×. (B) Representative scatter plots of FACS analysis for determining macrophages that engulfed apoptotic cells in each treatment group. (C) Quantitative analysis of efferocytotic ability of macrophages in each treatment group with a flow cytometry—based method. Values are mean ± SEM and represent average of findings from two independent experiments with triplicate samples in each group. **P < 0.01 versus the control group.