Transwell Assay for Macrophage Migration

The migration of the cultured macrophages was assessed by a transwell assay using 6.5 mm transwell chambers (8 µm pores, Corning Costar, New York, NY, USA, 3422) as described previously [25 (link),29 (link)]. After the chambers were pretreated with 0.1 mg/mL Poly-L-lysine hydrobromide (PLL, Sigma-Aldrich, St. Louis, MO, USA, P1274) solution or culture medium, 1 × 10⁵ macrophages in 100 µL of DMEM/F12 containing 1% FBS were seeded into the upper chamber, and the lower chamber was filled with 600 µL DMEM/F12 containing 10% FBS and 1 mg/mL myelin debris without cells. The macrophages were allowed to migrate for 18 h, and then the chambers were fixed with 4% PFA for 20 min. After careful removal of the cells on the upper surface with a cotton swab, the cells adhered to the lower surface of the transwell membrane were stained with 0.1% crystal violet (Leagene, Beijing, China, DZ0055) for 30 min. Then, five images of each membrane (the center and four quadrants) were captured under an inverted microscope (Leica, Wetzlar, Germany) for quantification.