Opadokun Tosin, Rohrbach Petra
Institute of Parasitology, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada.
Methods Protoc. 2024 Nov 9;7(6):92. doi: 10.3390/mps7060092.
Over the last few decades, malaria-derived extracellular vesicles (EVs) have gained increasing interest due to their role in disease pathophysiology and parasite biology. Unlike other EV research fields, the isolation of malaria EVs is not standardized, hampering inter-study comparisons. Most malaria EV studies isolate vesicles by the "gold-standard" technique of differential (ultra)centrifugation (DC). Here, we describe in detail an optimized and reproducible protocol for the isolation of malaria-derived EVs by DC. The protocol begins with a description of cultivating high-parasitemia, synchronous cultures that are the source of EV-containing conditioned culture media. The isolation protocol generates two EV subtypes, and we provide details of characterizing these distinct subtypes by analyzing human and parasite proteins by Western blot analysis. We identify some of these proteins as suitable markers for malaria EV subpopulations and subtypes.
在过去几十年中，源自疟疾的细胞外囊泡（EVs）因其在疾病病理生理学和寄生虫生物学中的作用而越来越受到关注。与其他EV研究领域不同，疟疾EVs的分离方法尚未标准化，这阻碍了研究间的比较。大多数疟疾EV研究通过差速（超速）离心（DC）这一“金标准”技术来分离囊泡。在此，我们详细描述一种通过DC分离疟疾来源EVs的优化且可重复的方案。该方案首先描述了培养高疟原虫血症的同步培养物，这些培养物是含EV的条件培养基的来源。分离方案产生两种EV亚型，我们通过蛋白质免疫印迹分析人类和寄生虫蛋白，提供了表征这些不同亚型的详细信息。我们将其中一些蛋白质鉴定为疟疾EV亚群和亚型的合适标志物。
