Isolation of Adipose Tissue Macrophages

Adipose tissue macrophages were isolated as previously described.^{13 (link)} Briefly, whole adipose tissue samples were cut into small pieces, gently homogenized in RPMI using disposable tissue grinders (Fisher Brand), and filtered through 70μm cell strainers (BD Flacon, Bedford, MA) to obtain single cell suspension. Macrophages were isolated by dual density gradient (Histopaque -1077 and Histopaque-1119, Sigma Aldrich). Isolated cells were incubated with FACS permeabilization solution 2 (BD Biosciences) for 30 min at 37 C, then measured by flow cytometry for cells positive for both CD14 (BD Pharmingen), and hVEGF-A _total (which does not distinguish isoforms) (R&D systems).