Author response image 4. Myelin is internalized and routed to lysosomal compartments by brain endothelial cells.
(A) BECs were loaded with Atto 633-labeled human myelin for 24h and internalization of myelin particles was assessed by imaging flow cytometry. To determine internalization scores, a mask was designed based on the surface of BECs in the brightfield image. This mask was then eroded to exclude the cell membrane. The resulting mask was applied to the fluorescence channel. The internalization score, interpreted as a ratio of the intensity of the intracellular space versus the intensity of the whole cell, was calculated on this mask using the internalization feature of the Ideas v6.0 software (AMNIS Merck Millipore). Cells that have internalized antigens have positive scores, as depicted here for BECs. (B-G) Adherent brain endothelial cells were incubated with Atto-633-labeled myelin and 24h later, co-localization of myelin (Red) with early endosomal (EEA1, Green, upper panels) or endosomal/lysosomal (LAMP1, Green, lower panels) compartments was analyzed using CSLM. Nuclei were visualized with Hoechst (blue). Representative images of adherent brain endothelial cells with subcellular localization of Myelin with EEA1 (or LAMP1 (C). A magnification of indicated areas is shown in D B)+E. F+G. Histograms were created for a selected area (indicated by a line) using ImageJ software (NIH, USA). Histograms were created from each fluorochrome and overlays were made by the program. Histograms clearly show the lack of association of myelin with early endosomes but the enclosure of myelin within LAMP1 positive vesicles.