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Background information

Exosomes are extracellular vesicles released from living cells in an energy-dependent process. Exosomes are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell. Exosomes are secreted by many cell types

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into diverse body fluids such as blood, semen, urine, saliva, breast milk, ascites fluid, and cerebrospinal fluid. The main difference to other extracellular vesicles such as apoptotic vesicles or membrane vesicles is the endocytic origin of exosomes. Exosomes are released from intact cells after inward budding of multivesicular bodies and fusion with the plasma membrane. They have the same membrane orientation as the originating cell, i.e., displaying extracellular domains on their surface. CD9, CD63, and CD81 are three of the most-studied members of the tetraspanin protein family and can be used to isolate exosomes.

Detailed procedure

The isolation of exosomes or extracellular vesicles (EVs) is performed by positive selection using MicroBeads recognizing the tetraspanin proteins CD9, CD63, or CD81. First, EVs are magnetically labeled during a short incubation period. The labeled EVs are loaded onto a μ Column, which is placed in the magnetic field of a μMACS

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Separator. The magnetically labeled EVs are retained within the column, while the unlabeled vesicels and cell components run through the column. After removing the column from the magnetic field, the intact EVs can either be collected by elution with Isolation Buffer, or directly lysed in the column and the protein in the lysates can be analyzed, e.g., by Western blotting.