Figure 1.

Characterisation and in vitro properties of BM-Ectosomes. (A) Flow cytometric characterisation of bone marrow-derived ectosomes (BM-Ecto). CFSE-positive events (representing intact vesicles) were analysed for surface expression of Gr-1 and phosphatidylserine (PS; using annexin V) (upper lane, left to right). Controls are (lower lane, left to right): annexin buffer alone, CFSE threshold set on sonicated BM-Ecto previously stained with CFSE, BM-Ecto stained with annexin V and IgG1 isotype in PBS. FSC denotes forward scatter, SSC side scatter, respectively. Numbers indicate % positive BM-Ecto (B) Morphology of BM-Ecto (size bar 100 nm) and (C) monosodium urate (MSU) crystals (size bar 50 µm) as determined by transmission electron microscopy and light microscopy, respectively. (D) In vitro stimulation protocol. B6 peritoneal macrophages were primed with LPS for 10 h and subsequently stimulated with 100 µg/mL MSU for 4 h. BM-Ecto (1×10⁸ BM-Ecto /2×10⁶ macrophages) were given either prior (BM-Ecto+LPS>MSU) or after (LPS>BM-Ecto+MSU) LPS priming as outlined. Alternatively, PS-liposomes or control PC-liposomes were given instead of BM-Ecto. (E and G) IL-1β in cell culture supernatants determined by ELISA. n=4 per group. (F and H) Cell extracts (XT) and supernatants (SN) were analysed for the presence of NALP3, pro-IL-1β, IL-1β and active caspase 1 (p20 and p10) by western blot. Data in A, F and H are representative of three independent experiments. ***p<0.001. Mean±SEM is shown.