Figure 1

Quantification of myelin phagocytosis by using a pH-sensitive fluorescent dye. (A) Myelin was labeled with pHrodo and resuspended in PBS pH 7.4, 6, or 4. Fluorescent intensity was measured on a Varioskan Flash and increased in signal with decreasing pH. (B) THP-1 macrophages were incubated without (gray line) or with 12.5 μg pHrodo-labeled myelin (black line) for 6 and 24 hours, and measured with flow cytometry. Fluorescence increased over time as THP-1 macrophages phagocytosed myelin. Uptake of pHrodo-labeled myelin (red) by (C) THP-1 macrophages and by (D) primary human microglia. Microglia were stained with phalloidin (green) to visualize actin filaments. Insert shows a close-up of a phagocytosing microglia. (E) Percentage of cells that phagocytosed pHrodo-labeled myelin in (E). THP-1 macrophages and (F) primary human microglia over time measured with flow cytometry. Scale bar is 25 μm.