Quantification of M1 and M2 Macrophages

Cells in the SVF were incubated in Pharm Lyse (BD Biosciences) for 15 min at 4°C and resuspended in Pharmingen stain buffer (BD Biosciences). The cells were incubated with 2.4G2 (BD Biosciences) for 10 min and then with primary antibodies or the matching control isotypes for 30 min at 4°C. Then, the cells were rinsed twice and resuspended in Pharmingen stain buffer. After incubating with 7-amino-actinomycin D (BD Biosciences), the cells were analyzed using a FACSAria cell sorter (BD Biosciences). The data analysis was performed using FlowJo (Tree Star, Ashland, OR). M1 or M2 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative or F4/80-positive/CD11c-negative/CD206-positive cells, respectively. The numbers of M1 or M2 macrophages were calculated by multiplying the number of trypan blue–negative cells by the ratio of F4/80-positive/CD11c-positive/CD206-negative cells or that of F4/80-positive/CD11c-negative/CD206-positive cells.