Exosomes are bound by CD169⁺macrophages in the spleen and LN in the absence of blood or lymph flow. Exo-bio was applied to naïve C57BL/6 or CD169^−/− spleen and LN sections using a modified Stamper-Woodruff assay. Biotin was detected with (A) streptavidin-Alexa-488 (green) or (B) -Alexa-594 (red) and marginal metallophilic or subcapsular sinus macrophages stained with anti-CD169 (MOMA-1). Primary antibody was detected with (B) anti-rat IgG-Alexa-488 (green) and nuclei counterstained with DAPI (blue). (C) Exo-bio ± treatment with Vibrio cholerae–derived sialidase (α2,3-linked sialic acid preferential cleavage [SIAL-V]) were purified using a sucrose cushion, negatively stained and visualized by transmission electron microscopy. No apparent differences in morphology were observed between samples; diameter range was 70 to 120 nm. Photographs are representative of the preparations as a whole. (D) Exo-bio was bound to naïve C57BL/6 sections in the presence of negative control antibody or CD169 neutralizing antibody (SER-4). Alternatively, Exo-bio or SIAL-V–treated Exo-bio was applied to naïve C57BL/6 or CD169^−/− sections, respectively. Exosomes and nuclei were detected as described in A. (E) Exosomes ± SIAL-V treatment was bound to aldehyde-sulfate microspheres and analyzed by flow cytometry for α2,3- and α2,6-linked sialic acid expression using biotinylated MAL-II and SNA lectins, respectively. In addition, CD9, CD24, MHC-II, CD19, immunoglobulin, and CD21 expression was measured. Shaded peak, negative control (BSA-conjugated aldehyde-sulfate microspheres); black line, untreated exosomes; dashed line, SIAL-V–treated exosomes. Results representative of ≥3 separate experiments and/or exosome preparations, with (D:LN) LN sections from ≥4 anatomically distinct locations per experiment. Original magnification, (A [spleen]) ×40, (A [LN]) ×100, (B,D) ×200, (C [Exo-bio]) ×24 500, and (C [SIAL-V–treated Exo-bio]) ×17 500. Bar represents (A [spleen]) 500 µm, (A [LN]) 250 µm, (B,D) 200 µm, (C) 500 nm, and (C, inset) 100 nm.