For the assay, wound macrophages were seeded in 8-well chambered slides. Apoptotic (5 µM dexamethasone treated for 12 h; yield >90% PS positive thymocytes, Fig. 3A ) thymocytes were added to each chamber in a (1∶10) macrophage:thymocyte ratio. Prior to co-culture with macrophages, thymocytes were labeled with a fluorescence cell-tracker reagent (CellTracker™ Orange CMTMR, Molecular Probes). Thymocytes have been largely used and are well accepted for efferocytosis studies performed using cultured macrophage ex vivo. Moreover, upon induction of apoptosis, both PMN and thymocytes are known to externalize phosphatidyl serine (PS), one of the key mechanisms of apoptotic cell recognition by macrophages [74] (link), [75] (link). Phagocytosis assay was performed for 1 h at 37°C. In co-culture studies, shorter incubation times (10–15 min) were used for adherence assay while longer (45–60 min) co-culture period were utilized for the phagocytosis assays [51] (link). Macrophages were then extensively washed to remove non-phagocytosed cells. Cells were fixed with 4% paraformaldehyde and stained using F4/80-FITC. Imaging was performed using a fluorescence microscope (thymocytes, red; macrophage green or phase contrast). Quantitation of phagocytosed thymocytes by each macrophage was performed using Axiovision software (Zeiss) by counting 50–100 macrophages from each well. Data are expressed as “phagocytic index”. This index is defined as the total number of apoptotic cells engulfed per macrophage present in the field of view [62] (link). This approach enables normalization of the data against macrophage number.
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