Variomacs technique

The human mononuclear cells were separated from the peripheral blood of healthy blood donors by utilizing Ficoll‐Paque (Amersham Biosciences, Sunnyvale, CA, USA). From the peripheral mononuclear cells, the CD14⁺ cells were isolated using VarioMACS™ technique and anti‐CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), through high‐gradient magnetic sorting. Macrophages were then induced from CD14⁺ monocytes for 6 days, which were cultured in complete RPMI‐1640 medium with 10 ng/ml human macrophage colony‐stimulating factor (M‐CSF) (R&D System, Minneapolis, MN, USA). Curcumin (Sigma, St. Louis, MO, USA) was dissolved in ethanol at a concentration of 10 mmol/L as stock solution. It was further diluted in phosphate‐buffered saline (PBS, vehicle) for use.

For human macrophage preparation, peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy human donors by standard density-gradient centrifugation with Ficoll-Paque (Amersham Biosciences). After centrifugation, the buffy coat containing leukocytes (PBMC) and platelets was further washed with PBS, and CD14⁺ cells were purified using the VarioMACS technique with anti-CD14 microbeads (Miltenyi Biotec GmbH). Cells were then cultured in complete RPMI 1640 medium supplemented with 10 ng/ml human M-CSF (R&D Systems) for 6 days^{4 (link)}. For preparation of murine bone marrow-derived macrophages, bone marrow cells were isolated from femurs and tibias and cultured in RPMI 1640 complete medium supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/ml of recombinant mouse M-CSF (R&D Systems) for 6–8 days. Human monocytes were obtained from healthy donors at the Taipei Blood Center of the Taiwan Blood Services Foundation, under a protocol (AS-IRB02-103202) approved by the IRB of the Clinical Center of the Department of Health, Taiwan. Written informed consent was obtained from all donors.