Comparison of microglia-decontamination methods. (A) Flow-cytometric assessment of enrichment of microglia in primary astrocyte cultures. Microglia (CD45int/CD11bposi/Gr-1nega-int) were not observed in primary astrocyte cultures after exposure to liposomal clodronate. (B) Immunostaining using Iba1 (red) and GFAP (green) antibodies. Scale bar, 50 μm. (C) The number of CD45-positive microglia in primary astrocyte cultures determined by FACS analysis (n = 6). (D) Quantification of the number of Iba1-positive microglia to the total number of cells in primary astrocyte cultures (n = 6). (E, F) Comparison of the gene expression of microglial markers such as Iba1, Cx3cr1, and Itgam (also known to CD11b) using semi-quantitative (E) and qPCR (F, n = 6). (G) The viability of astrocytes after exposure to liposomal clodronate or flow cytometric purification. There were few PI-positive dead cells 24 h after exposure to liposomal clodronate, but a large number of astrocytes were positive for PI 24 h after flow cytometric purification. (H, I) Flow cytometric analysis (H) and immunostaining (I) of astrocyte cultures 42 days after exposure to liposomal clodronate. Scale bar, 100 μm. Data are presented as the mean ± SEM.