Cell culture
For macrophage differentiation, monocytes were cultured at 37°C with 5%
CO2 in 24-well plates (Costar) for 6 days in
supplemented RPMI medium (10% fetal bovine serum, 10nM glutamine, 100
μg/ml streptomycin, 100 U/ml penicillin) and 50 ng/ml GM-CSF for M1
macrophages, then incubated for 24 hours with IFN-γ (100 ng/ml) and LPS
(100 ng/ml). For MMe and M2 macrophages, monocytes were incubated for 6
days with M-CSF (50 ng/ml), and then incubated for 24 hours with 15mM
glucose (Sigma Aldrich), 10nM insulin (Sigma Aldrich) and 0.4mM
palmitate (Sigma Aldrich) to polarize to MMe, or with IL-4 (20 ng/ml) to
obtain M2 macrophages. Cells were harvested using EDTA and further
evaluation by flow cytometry was performed. Culture media were collected
for cytokine measures.