M csf

For macrophage differentiation and polarization, CD14⁺ monocytes (1 × 10⁶ cells/well) were isolated and characterized by flow cytometry as described previously¹⁵, ¹⁶ and seeded in RPMI medium (Sigma‐Aldrich) containing 1% Penicillin/Streptomycin, 1% glutamine, 1% Fungizone, and 10% fetal calf serum, supplemented with either 50 ng/mL human recombinant GM‐CSF or M‐CSF (PeproTech) for 7 days. For β‐oxidation assays, M‐CSF‐differentiated macrophages were polarized into an anti‐inflammatory phenotype with 100 ng/ml IL‐4 (Novartis) and 10 ng/ml M‐CSF for 2 days. To analyse pro‐inflammatory cytokine gene expression, M‐CSF‐differentiated macrophages were stimulated with 100 ng/ml LPS (E. coli 055:B5, #L4005, Sigma) and treated with vehicle control or Vorinostat (#10009929, Cayman Chemicals) for 24 h. ELISA to measure secreted IL12p40 was described previously.¹⁶ To determine mean cell size and number of adherent or nonadherent cells, a CASY automated cell counter (Omni Life Sciences) was used. For detachment, adherent macrophages were incubated with 300 µL Gibco™ TrypLE™ Select (10×) (Gibco, Life Technologies) for 15 min at 37°C shaking every few minutes and was gently removed with a cell scraper.