Isolation and Differentiation of Human Macrophages

Macrophages were obtained from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated by density gradient centrifugation (Ficoll 1.077 g/mL; Lympholyte, Cedarlane Laboratories Ltd., Uden, The Netherlands), diluted at 1 × 106 cells/mL in α-Minimal Essential Medium (α-MEM) (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS) (Euroclone, Siziano, Italy), 100 IU/mL penicillin, and 100 g/mL streptomycin and L-glutamine (Gibco Limited, Uxbridge, UK), and plated in 24-well Cell Culture Multiwell. To obtain fully differentiated human macrophages, the PBMCs were cultured for 15 days in the presence of 25 ng/mL recombinant human macrophage colony-stimulating factor (rh-MCSF) (Peprotech, London, UK). Culture medium was replaced twice a week. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂. Macrophages derived from DMD patients were treated with JWH-133 and AM630 when they were completely differentiated. After 24 h of treatment exposure, cells were harvested for protein extraction, and cell cultures supernatants were collected to perform the iron assay and to analyze the release of several pro- and anti-inflammatory cytokines by means of enzyme-linked immunosorbent assay (ELISA).