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. Author manuscript; available in PMC: 2017 Jun 15.
Published in final edited form as: J Immunol. 2016 May 18;196(12):5089–5100. doi: 10.4049/jimmunol.1502270

Figure 2. Macrophages are the primary source of MFG-E8 at wound-site in inflammatory phase.

Figure 2

A, Western blot analysis of MFG-E8 protein expression in wound-edge tissues from C57bl/6 mice on d0-10 post wounding. β-Actin was used as loading control. Representative blot from four independent experiments has been provided. Densitometry quantification of the Western blot data was performed. Data is presented as % change compared to d0 (skin). Data are expressed as mean ± SD (n=4); *p<0.05 compared to d0 (skin). B, Western blot analysis of MFG-E8 protein expression in bone marrow derived monocytes (BMDM) and d3 wound mϕ (ωmϕ). β-Actin was used as a loading control. Representative blot from three independent experiments have been provided. Densitometry quantification of Western blot data has been presented. Data are expressed as mean ± SD (n=3); *p<0.05 as compared to BMDM. C, Representative images of wound-edge tissue sections d3-10 post wounding from ROSA-LysM mice displaying abundance of wound mϕ (GFP+, green) at the wound-site peaked on d3 and 5 post-wounding. Most cells/tissue in these mice widely express cell membrane-localized red fluorescence, however, because of LysM driven cre recombinase only the cells of myeloid origin express membrane-localized green fluorescence. DAPI (blue, nuclear) staining was performed on wound tissue sections followed by fluorescence microscopy and imaging. Scale bar = 200μm. D, Representative images of d3 wound-edge tissue sections from ROSA-LysM mice stained with MFG-E8 (purple) to observe co-localization of MFG-E8 with green wound mϕ. The bar-graph represents the ratio of MFG-E8 that is contributed by macrophages (MFG-E8 co-localized with GFP positive macrophages) to the total MFG-E8 (purple) in field of view. The ratio of purple:green co-localization in confocal images was determined using automated unbiased co-localization module in FV10-ASW software (Olympus). Scale bar = 50μm. Bar-graph represents the fraction of MFG-E8 that is contributed by macrophages.