Microglial culture protocol and Aβ-mediated activation of two phenotypic microglia. A. The diagram represents the culture protocol for mixed glia and purified microglia. Mixed glia were cultured to DIV 7 and 16. Amoeboid microglia were detached from the mixed glial culture at DIV 7 and cultured in Neurobasal medium (NB) for 24 h (PAM). PAM was further incubated in medium containing the LADMAC-conditioned medium for 6 days and then cultured in NB for 24 h to produce DPM. B. Mixed glial cells at DIV 7 and DIV16 (a,b), and mixed glial cells at DIV16 were treated with 5 μM oAβ1-42 for 24 h (c, d). Microglia were immunostained with anti-Iba-1 (red in a, b), anti-CD11b (red in c, d) and anti-Aβ antibodies (green in c, d). Nuclei were stained using Hoechst33258 (blue). C. The morphology of PAM (a) and DPM (c) were observed with phase-contrast microscopy. PAM and the spiny processes of DPM indicated in the marked areas were magnified (b, d). D. The activation states of PAM (a) and DPM (c) were examined with anti-CD11b (red) and anti-CD68 (green) antibodies. The phase contrast images of each florescent image are presented (b, d). E. The representative images show internalized fibrillar Aβ25-35 (a, c) and FITC-conjugated oAβ1-42 (b, d) in both PAM and DPM. Internalized Aβ25-35 was detected with anti-Aβ antibody (green). Each experiment was performed in triplicate.