Isolation of Cardiac Macrophages

Highly pure macrophage populations were isolated from the infarcted area of the heart as previously described (3 (link), 16 (link)). Hearts were digested as described above. Macrophages were then selected by magnetic microbead separation by first removing Ly6G neutrophils (Ly6G microbeads, Miltenyi #130-120-337) and collecting the remaining CD11b + cells (CD11b microbeads, Miltenyi #130-126-725). Macrophages were then plated in 24-well Seahorse cultureware in RPMI + 0.1% FBS for 2 h for Seahorse experiments or in 6-well plates for RNA extraction and real-time PCR. The 2 h incubation period was previously established to obtain optimal RNA quality while maintaining the in vivo phenotype, as assessed previously (3 (link)). For some experiments, macrophages were pooled from different mice to generate enough cells for downstream assays.