Liposomal clodronate effectively eliminates microglia from primary astrocyte cultures. (A, B) Flow cytometric analysis of primary astrocyte cultures 3 days after exposure to free (A) or liposomal clodronate (B). The result indicated dramatic reduction in the CD11 positive (left) and CD45int/CD11bpositive/Gr-1nega-int (right) populations in a concentration-dependent manner. Complete microglial elimination was achieved using 2,000 μg/mL of free clodronate and 100 μg/mL of liposomal clodronate. (C) Triple immunostaining of primary astrocyte cultures 24 h after exposure to each clodronate using GFAP (blue), CD68 (green), and PI (red) antibodies. There were many PI and GFAP double-positive cells after exposure to free clodronate. Scale bar, 200 μm. (D) Quantification of the number of GFAP-positive cells three days after exposure to free or liposomal clodronate (n = 5). The number of astrocytes significantly decreased after exposure to free clodronate (*P <0.05, Kruskal-Wallis test with Dunn’s multiple-comparison test). (E) The duration of exposure to liposomal clodronate required for complete microglial elimination from primary astrocyte cultures. Flow cytometric analysis demonstrated that microglial elimination was achieved 12 h after exposure to 100 μg/mL of liposomal clodronate. Data are presented as the mean ± SEM.