PDF(Pubmed)
Abstract：
OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles.
RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.
RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.
摘要：
目的：细胞外囊泡（EV）已被证明在促进肿瘤发生中起关键作用。随着电动汽车研究的发展，隔离标准化很重要，质量控制,研究中的表征和验证方法，以及探索故障排除解决方案的可靠参考。因此,本研究说明的目的是从多种乳腺癌细胞系中分离EVs,并描述和执行国际细胞外囊泡学会EVs研究(MISEV)的最小信息列表所概述的验证方案.
结果：要隔离电动汽车，采用了两种技术：超速离心和尺寸排阻色谱。超速离心在我们手中产生更好的EV回收率，因此用于进一步验证。为了满足MISEV的要求，蛋白质定量，阳性（CD9，CD63，TSG101）和阴性（TGFβ1，β-微管蛋白）标记的免疫印迹，进行了纳米流式细胞术和电子显微镜检查。通过这些实验,我们证明了经过验证的EV的产量在不同的乳腺癌细胞系之间存在差异.方案进行了优化，以适应低水平的电动汽车，和各种技术和故障排除建议包括潜在的应用于其他细胞类型,可能为对未来EV研究感兴趣的研究人员提供益处.
结果：要隔离电动汽车，采用了两种技术：超速离心和尺寸排阻色谱。超速离心在我们手中产生更好的EV回收率，因此用于进一步验证。为了满足MISEV的要求，蛋白质定量，阳性（CD9，CD63，TSG101）和阴性（TGFβ1，β-微管蛋白）标记的免疫印迹，进行了纳米流式细胞术和电子显微镜检查。通过这些实验,我们证明了经过验证的EV的产量在不同的乳腺癌细胞系之间存在差异.方案进行了优化，以适应低水平的电动汽车，和各种技术和故障排除建议包括潜在的应用于其他细胞类型,可能为对未来EV研究感兴趣的研究人员提供益处.
