Protocol for isolating and characterizing extracellular vesicles by ultracentrifugation from bone marrow-derived macrophages.

Extracellular vesicles (EVs) enable the transmission of crucial molecular components between the parental and recipient cells. Macrophages can polarize into two distinct macrophage phenotypes, thereby exerting diverse effects on recipient cells. Here, we present a protocol for the direct isolation of EVs from bone marrow-derived macrophages (BMDMs) using ultracentrifugation. We describe steps for culturing BMDMs and macrophage polarization. We then detail procedures for further characterizing the isolated EVs using transmission electron microscopy (TEM), western blotting, and nanoparticle tracking analysis (NTA).