signature=e100a77f9e5994135d27edcec0fb2455,Extracellular vesicles released from chronic lymphocytic ...

Figure 1.

Characterization of extracellular vesicles. (A)…

Figure 1.

Characterization of extracellular vesicles. (A) Size distribution of CLL-derived vesicles (untreated, left panel,…

Figure 1.

Characterization of extracellular vesicles. (A) Size distribution of CLL-derived vesicles (untreated, left panel, or treated with a TLR9 agonist (CpG, right panel) analyzed by NanoSight Technology (NS300, Malvern). Mean size diameter of vesicles from untreated/treated samples was 106/124 nm for CLL1, 115/99 nm for CLL2 and 97/98 nm for CLL3. All samples were similarly diluted and measured with a sample flow rate of 10 (n=6). (B) Western blot analysis of the exosome-enriched proteins CD63 and CD9 (both antibodies from BioLegend). EV released by 5×109 CLL cells within 38–45 h were resuspended in 40 μL non-reducing lysis buffer; sodium dodecyl sulfate (SDS) gel electrophoresis was performed using 10 μL EV/lane in 12.5% SDS gels and subsequently proteins were transferred to a nitrocellulose membrane. (C) The amount of purified EV protein released by 108 cells was measured with Nanodrop1000 (Thermo Scientific) (n=11). (D) Flow cytometric analysis of CLL cells after 38–45 h culture in Panserin medium with or without 200 nM CpG. Most patients had IgHV mutations (see black circles or squares). Samples without mutations are represented by non-filled circles or non-filled squares. Cell viability was assessed with an apoptosis assay, staining with annexin V-PE and 7-AAD (BioLegend) (PS = phosphatidylserine). Cells were stained with CD69-FITC (BD Biosciences), CD25-FITC (BD Biosciences), purified CD54 and CD82 (both BioLegend), which were detected with goat-anti-mouse-PE (BioLegend). The analysis was performed with a FACS Calibur (BD Biosciences) or FACS Gallios (BeckmanCoulter). Dead cells were excluded by gating for 7AAD (BD Biosciences) negative cells (n=5–14). (E) Flow cytometric analysis of EV: EV were bound to polystyrene beads (Polysciences) and subsequently stained and analyzed as described in (D). Only EV derived from cell samples with a viability of more than 90% were used for further analysis (n=5–9). The IgHV status of one patient analyzed for CD54 expression (left panel) is unknown (labeled with an x). (F) One representative histogram for FACS data depicted in (E).