Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury

Extracellular vesicles (EVs) are small membranous particles that contribute to intercellular communications. Separating EVs from tissue is still a technical challenge. Here, we present a rigorous method for extracting EVs from intestinal tissue in a mouse intestinal ischemia/reperfusion (I/R) model, and for analyzing their miRNA content. The isolated EVs show a typical cup shape with a size peak of 120-130 nm in diameter, confirmed by TEM and NTA. They also express EV markers such as CD9, CD63, CD81, Tsg101 and Alix. Real-time qPCR confirmed that these pellets contain miRNAs related to I/R injury. Our study presents a practical way to isolate EVs from intestinal tissue which is suitable for downstream applications such as miRNA analysis, and provides a novel method for investigating the mechanism of intestinal I/R injury. METHOD SUMMARY We present a practical way to isolate EVs from intestinal tissue; the method consists of enzymatic digestion, differential centrifugation and density gradient centrifugation. This method is suitable for downstream studies such as miRNA analysis.