Evaluation of macrophage polarization by cell sorting and FACS analysis. (A) Representative FACS plots of muscle-infiltrating leukocytes stained for Ly6C and plotted against SSC for OPN^+/+mdx and OPN^−/−mdx. (B) Demonstration of the purity of MACS sorting. The top left FACS plot shows F4/80 staining of the negatively sorted population, and the top right FACS plot shows F4/80 staining of the positively sorted population. Positively sorted cells (arrow) were examined by SSC and FSC to isolate macrophages (larger population on the right) from eosinophils (left). The bottom right FACS plot shows Ly6C isotype control staining on the macrophage population used in these studies. (C) Representative FACS plots of MACS-sorted F4/80⁺ macrophages stained for F4/80 and Ly6C. Circled regions represent distinct macrophage populations. Experiments were done at 4 and 8 wk of age. Quantification of these experiments is shown in Fig. 4.