Figure 3. Dead cell clearance by wound-site macrophages.

For dead cell clearance assay, wound macrophages were co-cultured with cell-tracker labeled (red) thymocytes. A, Thymocyte apopotosis detected using Annexin V (FITC conjugated). Annexin V binds to externalized phosphatidyl serine (PS), a characteristics of apoptotic cells. Such treatment resulted in over 90% cells becoming phosphatidylserine (PS) positive. Data are mean ± SD; p<0.05 (n = 4). B, F4/80-FITC (green) and DAPI (blue, nuclear) stained wound macrophage establishing link with an apoptotic thymocyte (red); C, wound macrophage (F4/80-FITC and DAPI stained) engulfed a number of red apoptotic thymocytes; D, co-cultures of control (untreated, viable) thymocytes (red) with wound macrophage (DIC image) followed by wash; E, co-cultures of apoptotic thymocytes (red) with wound macrophages (differential image contrast, DIC image) followed by wash; F–G, Representative high magnification image of a macrophage in DIC (F) or stained with F4/80 FITC (green, G) showing engulfed and adhered (white arrows) apoptotic thymocytes (red). H, scoring of thymocytes engulfed by macrophage. Data are presented as phagocytic index which is defined as total number of apoptotic cells engulfed by macrophages in a field of view divided by total number of macrophage presented in the view. This approach enables normalization of the data against macrophage number. Data presented as mean ± SD (n = 3). *, p<0.01 compared to macrophage co-cultured with control thymocytes.