Macrophage Inflammatory Assay

Raw264.7 murine macrophages were suspended in DMEM and stimulated with 100 ng/mL lipopolysaccharide (LPS, Invitrogen). After 10 minutes, the stimulus was removed via centrifugation at 250g for 5 minutes and unstimulated or stimulated macrophages were plated at 25,000 cells/cm² in 12-well culture plates. CM from Spheroid 1 and Spheroid 2 was collected and diluted in α-MEM such that the ratio of growth factor-producing cells to CM volume was kept constant. The CM was then added to stimulated macrophages at a 1:10 dilution. After 24 hours of culture, the macrophage CM was collected and assessed for pro- and anti-inflammatory markers. PGE₂ (1 ng/mL, Sigma) was used as a positive control. The polarization of the macrophages was determined by measuring pro-inflammatory TNFα and anti-inflammatory IL-10 using mouse protein-specific ELISA kits (R&D Systems).