EV Purification and Analysis Core

摘要-核心2 细胞外囊泡纯化和分析（EVPA）核心将专门服务于三个单独的项目 在计划项目补助金中。总的来说，核心的目标是集中方法，专业知识，设备， 和熟练的劳动力来进行细胞外囊泡（EV）纯化、分析、大小测定、计数和质量控制。 Core将通过差速离心和密度梯度超离心分离EV，使用Optiprep/ 碘克沙醇分层。将使用纳米颗粒跟踪分析对EV进行计数和大小测定。此外，该核心将 进行基于荧光团缀合抗体的荧光激活囊泡分选（FAVS）， 标记EV表面标记和遗传编码荧光标记以标记内部蛋白。这种技术 允许对自然低于光的衍射极限的EV进行选通（计数）和分选， 尺寸EV制剂将基于专门的密度梯度进一步细分以追踪EV蛋白 标记和RNA含量以及分离的外泌体。对于项目1，该核心将净化电动汽车的所有3个目标，以及 为目标1C提供EV细分和FAVS EV分析和分类。对于项目2，该核心将净化 电动汽车的所有3个目标（1-3）。对于项目3，该核心将利用FAVS EV分析和分选以及EV和exomere 纯化以实现所有3个目的（1-3）的目标。EVPA核心还将从事技术开发 进一步开发FAVS方法，以基于荧光RNA和RBP分析单个EV，并使用 固定的、透化的EV用于分析。 总而言之，核心的目标是为所有3个方面提供统一的纯化和分析实践。 项目和独特的熟练技术人员和设备集中在一个核心。因此，这一结构将有助于 广泛但及时的表征和交付高质量的材料给调查人员，并推动切割 在这个领域的边缘。

SUMMARY - Core 2 The Extracellular Vesicle Purification and Analysis (EVPA) Core will uniquely serve the three individual projects within the Program Project Grant. Overall, the goal of the Core is centralize the methods, expertise, equipment, and skilled labor to perform extracellular vesicle (EV) purification, analysis, sizing, counting, and quality control. The Core will isolate EVs by differential centrifugation and density-gradient ultracentrifugation using Optiprep/ iodixanol layering. EVs will be counted and sized using nanoparticle tracking analysis. Moreover, this Core will perform Fluorescent Activated Vesicle Sorting (FAVS) which is based on fluorophore-conjugated antibodies to label EV surface markers and genetically encoded fluorescent markers to label internal proteins. This technique allows for the gating (counting) and sorting of EVs which are naturally below the diffraction limit of light based on size. EV preparations will further be sub-fractionated based on specialized density gradients to trace EV protein markers and RNA contents and isolate exomeres. For Project 1, this Core will purify EVs for all 3 Aims, as well as provide EV subfractionation and FAVS EV analysis and sorting for Aim 1C. For Project 2, this Core will purify EVs for all 3 Aims (1-3). For Project 3, this Core will utilize FAVS EV analysis and sorting and EV and exomere purification to achieve the goals of all 3 Aims (1-3). The EVPA Core will also engage in technology development to further develop the FAVS method to analyze single EVs based on fluorescent RNAs and RBPs, and to use fixed, permeabilized EVs for analysis. In summary, the goals of the Core are to provide uniform purification and analysis practices for all 3 projects and uniquely skilled technicians and equipment in a centralized Core. As such, this structure will facilitate extensive but timely characterization and delivery of high quality materials to investigators and drive the cutting edge in this area.