Extracellular Vesicles Containing Ceramide-Rich Platforms: "Mobile Raft" Isolation and Analysis

Extracellular vesicles (EVs) are secreted by eukaryotic cells and serve as carriers for a variety of cell signaling factors, including RNAs, proteins, and lipids. We described a unique population of EVs, the membrane of which is highly enriched with the sphingolipid ceramide. We suggested that ceramide in the EV membrane is organized in ceramide-rich platforms (CRPs), a type of lipid raft that mediates interaction of ceramide with ceramide-associated proteins (CAPs). Here, we describe methods using anti-ceramide antibody to isolate ceramide-enriched EVs and detect exosomes after uptake into recipient cells. In addition, we discuss methods for EV analysis using nanoparticle tracking and mass spectrometry. The methods can be extended to the isolation of other types of EVs and "mobile rafts" transported by EVs from donor to recipient cells using antibodies against lipids specific for these EVs.