Characterization of human myelin. (a) Myelin was prepared by homogenization of fresh human white matter and sucrose gradient centrifugation, as indicated in the scheme. (b) The resulting myelin particles were characterized by FACS using microglial (MOG and PLP), endothelial (CD31), microglial (MHC-II), astrocytic (GFAP) or neuronal (NGFR) markers (representative of n > 3 donors). (c) Transmission electron micrograph of myelin particles (representative of n > 3 donors). (d) The diameter of 100 myelin particles was measured in 10 different micrographs. Data are the mean ± SD (n = 3 independent experiments). (e) Myelin was coated on to ELISA plates and the binding of DC-SIGN measured by a DC-SIGN–binding assay as described in the Materials and methods. *, P ≤ 0.05 (Mann-Whitney U test). Data are the mean ± SD (n = 3 independent experiments). (f) Binding of a titration of atto633-labeled myelin to DC-SIGN–expressing DCs was measured by FACS. Data are the mean ± SD (n = 3 independent experiments). (g) DCs were incubated with myelin (10 µg/ml), the DC-SIGN ligand PAA-LeX (10 µg/ml), or the MGL-ligand PAA-GalNAc (10 µg/ml), washed, and fixated. Binding of the DC-SIGN–specific antibodies AZN-D1 (mAb against carbohydrate-recognition domain) and CSRD (polyclonal against C terminus) was determined by FACS. Data are shown as the mean ± SD (n = 3 independent experiments).
