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GLIA_22337_sm_SuppFig1.pdf99KSupporting Information Figure 1. Inhibition of PKD reduced dextran macropinocytosis. (A) The cells were stimulated with UDP (100 µM) in the presence of FITC-dextran and β-amyloid. After washing off by HBSS, the images were obtained using a confocal microscopy. The fluorescence of dextran and β-amyloid was seen in the same vacuoles. (B) The cells were stimulated with UDP (100 µM) in the presence of IgG-opsonized microspheres and β-amyloid. After washing off, the images were obtained using a confocal microscopy. The fluorescence of IgG-opsonized microspheres and β-amyloid was seen in the same vacuoles.
GLIA_22337_sm_SuppFig2.pdf107KSupporting Information Figure 2. Mouse spinal cord slices (300 µm thickness) were obtained from mouse expressing EGFP under iba1 promoter which received uni-lateral peripheral nerve (L4 spinal nerve) transaction 7 days before slice preparation. After 30 min incubation in HBSS for recovery, Slices were incubated with 2 µM β-amyloid-Hilyte555 and 100µM UDPβS (BIOLOG, Bremen, Germany) at 37°C. The Slices were placed on the glassbottom dish, then conforcal image was obtained using CellVoyager CV1000 (YOKOGAWA, Japan), temperature was maintaitned at 37°C. Observation ROI was placed in dorsal spinal cord in which microglia were enriched by nerve injury. Formation of vacuoles in the EGFP expressing cells was detected by their morphology and particles of β-amyloid-derived fluorescence in the vacuoles were counted and plotted. Primary microglial culture was obtained from adult rat (10 weeks old) brain cortex. Rat was anesthetized by intraperitoneal pentobarbital injection and transcardially perfused by ice cold PBS(-). Brain cortex was removed and minsed by scalpel. Cell suspension was obtained using Neural Tissue Dissociation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). Microglial cell were isolated by percoll density separation according to previous report (Alexander H et al., Glia, 2007, 55:1374-1384). Cells were cultured in glassbottom dishes in DMEM supplemented with 10% FBS 30 ng/mL rat recombinant GM-CSF for 5days. Fluorescent incorporation assay was performed as the same way in MATERIAL and METHODS.
GLIA_22337_sm_SuppVideo1.avi4119KSupporting Information Video 1. Cells were plated onto a poly-L-lysine coated glass-bottom dish and differential interference contrast images were acquired using a confocal microscope (LSM510; Carl Zeiss, Jena, Germany) at room temperature. During observation, culture medium was replaced with Hank's balanced salt solution (HBSS) supplemented with 10 mM HEPES (pH 7.4). Glass microcapillaries, with 0.5 inner diameters, were filled with 1 mM UDP and positioned near cells with a manipulator without any pressure into the capillary.
GLIA_22337_sm_SuppVideo2.avi4336KSupporting Information Video 2. Cells were plated onto a poly-L-lysine coated glass-bottom dish and differential interference contrast images were acquired using a confocal microscope (LSM510; Carl Zeiss, Jena, Germany) at room temperature. During observation, culture medium was replaced with Hank's balanced salt solution (HBSS) supplemented with 10 mM HEPES (pH 7.4). Glass microcapillaries, with 0.5 inner diameters, were filled with 1 mM UDP and positioned near cells with a manipulator without any pressure into the capillary.

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