Immunostaining of extracellular vesicles (EVs) has become necessary for the characterization of EV subtypes, clarification of the EV biogenesis/cellular uptake pathway, drug delivery, etc. Immunostained EVs must be in suspension for further downstream analyses or uses. However, conventional EV immunostaining methods yielding EVs in suspension lack either sufficient recovery or staining specificity because of the washing steps. In this study, we have devised and tested a method to wash immunostained EVs with successive aqueous two-phase system (ATPS) separations. The ATPS is a liquid-liquid extraction procedure that ensures a gentle separation of target molecules. The ATPS has been successfully employed to separate EVs from other impurities with high yield and high purity. Immunostained EVs were washed with the ATPS and compared with other immunostaining methods to confirm the proposed method's high EV recovery and staining accuracy. According to the result, the ATPS-based EV immunostaining method required as low as ∼1 μg without compromise of accuracy and recovery.
Keywords: aqueous two-phase system; biomolecule purification; extracellular vesicle; immunostaining; molecular characterization; tetraspanin analysis.