Macrophage activation by HMGB1 peptide

Bone-marrow cells collected from the femurs of 10-week-old male mice were cultured at 37 °C in the presence of 5% CO₂ in ultra-low attachment flasks (Corning, Corning, NY, USA) and medium (Dulbecco’s modified Eagle’s medium/F12; Thermo Fisher Scientific) containing 20 ng/mL macrophage colony-stimulating factor-1 (Peprotech Inc., Rocky Hill, NJ, USA); the medium was changed twice weekly, as described previously [6 (link)]. After 7 days, the macrophages were harvested. The harvested macrophages were seeded in 6-well ultra-low attachment dishes (Corning) at a density of 5 × 10⁶ cells/well. Then, 0.1 mg HMGB1 peptide (concentration: 1 mg/mL), 0.01 mg HMGB1 peptide (concentration: 0.1 mg/mL, low-dose group), or NS (control group) was added to the cultured macrophages. After 48 h, macrophages were harvested, and mRNA expression levels of genes encoding pro-inflammatory factors (e.g., interleukin-6 [Il6], tumor necrosis factor [Tnfa], monocyte chemotactic protein-1 [Mcp1], and inducible nitric oxide synthase [Inos]) and anti-inflammatory factors (e.g., Il-10, chitinase 3-like 3 [Ym1], found in inflammatory zone protein [Fizz1], and Cd206) were evaluated using real-time polymerase chain reaction (PCR).