2.8. Transwell migration assay
Transwell migration analysis was performed in 24-well Boyden chamber (diameter 6.5mm, pore diameter 8.0μm, CORNING). Mouse peritoneal macrophages were isolated by time difference adhesion method, and they were treated with the number of 1×104/well was seeded in the upper chamber. The macrophages in the upper chamber were treated by recombinant HMGB1 A Box protein to the final concentration of 200ng/ml or AMD3100 to the final concentration of 200ng/ml or CXCR4 neutralizing antibody to the final concentration of 5ug/ml; Primary cells from the midbrain with different treatments or combinations were placed in the lower chamber. After 24 hours of co-culture, the cells on the inner side of the upper chamber were gently removed. The cells on the lower side of the chamber were fixed with 4% paraformaldehyde, then stained with crystal violet, photographed under the microscope with 200 times magnification, and counted in 3 random fields with 400 times magnification.