EV Purification and Analysis Core

EV净化与分析核心

基本信息

批准号：
10544819
负责人：
Alissa M Weaver
金额：
$ 24.3万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-22 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10544819
关键词：
Area Biochemical Biogenesis Cell Culture Techniques Cell membrane Cells Centrifugation Conditioned Culture Media Cost Analysis Culture Media Dedications Density Gradient Centrifugation Development Doctor of Philosophy Endosomes Equipment Fluorescence Future Goals Heterogeneity Individual Knowledge Label Laboratories Large Intestine Carcinoma Left Light Membrane Methodology Methods Molecular Participant Permeability Population Population Heterogeneity Preparation Procedures Production Productivity Program Research Project Grants Property Proteins Quality Control RNA RNA analysis Reagent Reporting Research Research Personnel Resources Sampling Services Site Sorting Speed Standardization Structure Surface Technical Expertise Techniques Testing Training and Education Vesicle Work antibody conjugate biomarker development cost density design exosome experimental study extracellular vesicles fluorophore innovation instrumentation iodixanol meetings microvesicles nanoparticle novel particle program costs programs protein biomarkers skills technology development vesicular release

项目摘要

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.

摘要 - 核心 2 细胞外囊泡纯化和分析 (EVPA) 核心将为这三个单独的项目提供独特的服务在计划项目拨款范围内。总体而言，核心的目标是集中方法、专业知识、设备、以及执行细胞外囊泡 (EV) 纯化、分析、大小测定、计数和质量控制的熟练劳动力。 Core 将使用 Optiprep/ 通过差速离心和密度梯度超速离心分离 EV 碘克沙醇分层。将使用纳米颗粒跟踪分析对电动汽车进行计数和尺寸确定。此外，该核心将执行基于荧光团偶联抗体的荧光激活囊泡分选 (FAVS) 标记 EV 表面标记和基因编码荧光标记来标记内部蛋白质。这种技术允许对自然低于光衍射极限的 EV 进行选通（计数）和分类尺寸。 EV 制剂将根据专门的密度梯度进一步细分，以追踪 EV 蛋白标记和 RNA 含量并分离外显子。对于项目 1，该核心还将净化电动汽车以实现所有 3 个目标为 Aim 1C 提供 EV 细分和 FAVS EV 分析和分类。对于项目 2，该核心将净化所有 3 个目标 (1-3) 的 EV。对于项目 3，该核心将利用 FAVS EV 分析和分类以及 EV 和外显子净化以实现所有 3 个目标 (1-3)。 EVPA Core也将从事技术开发进一步开发 FAVS 方法来分析基于荧光 RNA 和 RBP 的单个 EV，并使用用于分析的固定、透化 EV。总之，核心的目标是为所有 3 个领域提供统一的纯化和分析实践集中核心中的项目以及独特技能的技术人员和设备。因此，这种结构将有利于广泛而及时地表征并向研究人员提供高质量材料并推动切割在这个区域的边缘。