PBMCs were isolated using density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Monocytes isolated by plastic adherence were differentiated into macrophages in RPMI 1640 medium (Life Technologies) supplemented with 20 ng/ml of M-CSF (eBioscience) and 10% of FBS (Lonza) for 5 d. Alternatively, monocytes were isolated using the EasySep human monocyte enrichment kit without CD16 depletion (STEMCELL Technologies). Macrophages were further differentiated into M1 or M2 macrophages by stimulation with 100 U/ml of IFN-γ (Sino Biologicals) and 100 ng/ml of LPS (Sigma-Aldrich), or 10 ng/ml of IL-4 and 10 ng/ml of IL-13 (Sino Biologicals). Attached macrophages were dissociated from plates using StemPro Accutase Cell Dissociation Reagent (Life Technologies).
To scavenge ROS, macrophages were stimulated in the presence of the ROS-scavenger Tempol (Sigma-Aldrich) or the mtROS scavenger MitoTempo (Santa Cruz Biotechnology, Inc.). Assembly of the NOX2 membrane complex was inhibited with gp91dstat (Anaspec). Glycolytic activity was blocked with 10 mM of 2-DG or by using glucose free RPMI 1640 medium (Life Technologies). PKM2 tetramerization was enforced with 50 µM of ML265 (Cayman Chemical). HIF-1α was inhibited with 10 µM of CAS (934593–90-5; Santa Cruz Biotechnology, Inc.). STAT3 phosphorylation was inhibited with 5 µM of Stattic (Sigma-Aldrich).
To scavenge ROS, macrophages were stimulated in the presence of the ROS-scavenger Tempol (Sigma-Aldrich) or the mtROS scavenger MitoTempo (Santa Cruz Biotechnology, Inc.). Assembly of the NOX2 membrane complex was inhibited with gp91dstat (Anaspec). Glycolytic activity was blocked with 10 mM of 2-DG or by using glucose free RPMI 1640 medium (Life Technologies). PKM2 tetramerization was enforced with 50 µM of ML265 (Cayman Chemical). HIF-1α was inhibited with 10 µM of CAS (934593–90-5; Santa Cruz Biotechnology, Inc.). STAT3 phosphorylation was inhibited with 5 µM of Stattic (Sigma-Aldrich).
