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.
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.