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.