Analysis of exosomes secreted by Daudi cells. (A) Exosomes were obtained by differential centrifugation as described in “Materials and methods” and deposited on a linear sucrose gradient. Fractions were collected and analyzed by Western blot and dot blot for the indicated markers. Indicated densities (grams per milliliters) were obtained for each fraction by refractometry. (B) Alternatively, exosomes were immunoadsorbed on the surface of magnetic beads coated with anti-HLA DR (clone BL2) and processed for electron microscopy (EM) as described in “Materials and methods.” (C) FACS analysis of beads coated with different membrane or subdomains from Daudi cells. Latex beads were coated with exosomes (Exos), or with LDTI membranes isolated from cells (LDTI) or from secreted vesicles (LDTI from exos) as described in “Materials and methods.” Membrane-coated beads were then analyzed for the presence of the ganglioside G_M1 through bCTXB binding and PE-streptavidin detection (FL-2, upper panel), and for the presence of MHC class II molecules through anti-HLA-DR antibody binding and FITC-antimouse IgG detection (FL-1, lower panel). CTRLs are beads incubated with PE-streptavidin but without bCTXB (FL-2, upper panel) and beads incubated with FITC-antimouse IgG but without anti-HLA-DR (FL-1, lower panel). (D) The same membrane fractions were immunoadsorbed on magnetic beads through the MHC class II molecules as described in “Materials and methods” and analyzed for the presence of G_M1 as in panel C. CTRL represent beads incubated with PE-streptavidin without bCTXB.