Kosanović Maja, Milutinović Bojana, Goč Sanja, Mitić Ninoslav, Janković Miroslava
Institute for the Application of Nuclear Energy, INEP, University of Belgrade, Belgrade, Serbia.
Biotechniques. 2017 Aug 1;63(2):65-71. doi: 10.2144/000114575.
Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.
尽管进行了大量研究，但事实证明，分离细胞外囊泡（EVs）的纯制剂具有挑战性。在此，我们将离子交换色谱法（IEC）与广泛使用的蔗糖密度梯度（SDG）离心法用于EVs的纯化进行了比较。通过差速离心，随后进行IEC或蔗糖密度梯度分离，从汇集的正常人羊水（AF）中分离出大量的EVs。通过电泳和凝集素印迹/免疫印迹评估分离出的EVs的纯度，以监测总蛋白、不同EVs标志物和选定的N-聚糖的分布。我们的数据显示，带负电荷的EVs能与其他带不同电荷的分子有效分离，而使用SDG离心对EVs进行的比较分析证实，阴离子交换色谱法有利于EVs的纯化。最后，尽管这种基于IEC的方法是使用AF进行验证的，但该方法应该也很容易适用于从其他来源分离EVs。
