Macrophage Differentiation from THP-1 Cells

The human monocytic cell line, THP-1, was obtained from ECACC and used for this study between passages 7 and 35. THP-1 cells were cultured in R10 medium composed of RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Massachusetts, Grand Island, NY, USA) and 2 mM L-glutamine, in the absence of exogenously added antibiotics. Pro-inflammatory (M1-like) macrophages and anti-inflammatory (M2-like) macrophages were generated by differentiation of these monocytes in the presence of 25 ng/mL phorbol 12-myristate 13-acetate (PMA) for three days or 10 nM 1,25-(OH)₂-Vitamin D₃ (Sigma-Aldrich, Poole, UK), for seven days, respectively [33 (link)]. M1-like macrophages were then washed and allowed to rest in fresh R10 medium without PMA for an additional 24 h [34 (link)]. Macrophages were plated out to a final density of 5 × 10⁵ cells/500 µL/well in R10 medium in 24 flat-bottomed well tissue culture plates.