The contents of EVs may potentially be useful as cell-type specific biomarkers 5, 6, 7, 8. Recent studies have shown that EVs act as messengers of intercellular communication networks, allowing cells to transfer lipids, proteins and RNAs between the producing cells and target cells 3, 4. EVs consist of a heterogeneous population of exosomes (30–100 nm in diameter) of endocytic origin and microvesicles (100–1000 nm in diameter), shed fragments of plasma membrane. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.Įxtracellular vesicles (EVs) are small membrane vesicles, composed of a lipid bilayer with inserted transmembrane proteins, enclosing cytosolic components (protein, mRNA and miRNA) derived from the EV-producing cells 1, 2. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes.
Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. Because the binding is Ca 2+-dependent, intact EVs can be easily released from Tim4 by adding Ca 2+ chelators. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. EVs carry lipids, proteins and RNAs derived from their producing cells and have potential as biomarkers specific to cell types and even cellular states. Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells.
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