Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Gyeong-buk 790-784, Republic of Korea.
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeong-buk 790-784, Republic of Korea.
ACS Appl Bio Mater. 2021 Apr 19;4(4):3294-3303. doi: 10.1021/acsabm.0c01625. Epub 2021 Mar 10.
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.
免疫染色法已成为鉴定细胞外囊泡（EVs）亚型、阐明 EV 生物发生/细胞摄取途径、药物传递等方面的必要手段。免疫染色的 EVs 必须悬浮在溶液中，以便进行进一步的下游分析或应用。然而，由于洗涤步骤的原因，传统的 EV 免疫染色方法得到的悬浮 EVs 要么回收率不足，要么染色特异性差。在本研究中，我们设计并测试了一种使用连续双水相系统（ATPS）分离来洗涤免疫染色的 EVs 的方法。ATPS 是一种液-液萃取程序，可确保目标分子的温和分离。ATPS 已成功用于从其他杂质中以高产率和高纯度分离 EVs。我们用 ATPS 洗涤免疫染色的 EVs，并与其他免疫染色方法进行比较，以确认所提出方法具有高 EV 回收率和染色准确性。结果表明，基于 ATPS 的 EV 免疫染色方法所需的起始量低至约 1μg，而不会影响准确性和回收率。
