Figure 2.

In vitro assays studying engulfment of synaptosomes and neural progenitor cells (NPCs) using human microglia-like cells (hiMG) from two subjects, human fetal primary microglia (MGf) from one subject, human monocyte-derived macrophages (MΦ) from two subjects and a human SV40 immortalized microglia cell line (imMG). (a, b) Representative imaging real-time live images in phase-contrast/red fluorescence mode (a) and in red fluorescence mode (b) of pHrodo (red)-labeled synaptosomes uptake in hiMG after 5 h. (c) Quantification of pHrodo (red)-labeled synaptosomes uptake (live imaging; 5 h). hiMG vs MΦ P<0.0001, hiMG vs MGf; P=NS. (d) Quantification of pHrodo (red)-labeled synaptosomes uptake (confocal microscopy). hiMG vs MΦ P<0.001, hiMG vs MGf; P=NS. See Supplementary Figure S7 for representative confocal images. (e) Uptake in hiMG (5 h) of pHrodo (red)-labeled NPCs using confocal microscopy. hiMG vs MΦ P<0.0001, hiMG vs MGf; P=NS, hiMG vs imMG; P=0.0001. See Supplementary Figure S8 for representative confocal images. (f) Relative uptake of pHrodo (red)-labeled synaptosomes (5 h) in hiMG with and without 30 min of anti-α_M (CD11b) M1/70 antibody pre-treatment as well as with pre-treatment using an isotype control antibody. Data are normalized to 'no antibody' (confocal microscopy). (g) hIMG uptake of pHrodo (red)-labeled synaptosomes isolated from NPC-derived neuronal cultures vs brain after 5 h (confocal microscopy; experiments controlled by total protein amount added). (h) Ratio of PSD-95 and phRodo immunoreactivity in hiMG treated with synaptosomes (5 h) isolated from NPC-derived neuronal cultures vs brain (confocal microscopy). (i, j) Representative confocal images of hiMG treated with cultured synaptosomes (i) and with synaptosomes isolated from brain (j). Scale bar=60 μm. Error bars represent s.e.m. Phagocytic index represents total uptake area of puncta (0.5–1.5 μm) located in cell area and divided on cell area. All P-values are two-sided. ^***P<0.01, ^****P<0.0001.