使用成像流式细胞术表征从细胞培养基，血浆或尿液分离出的细胞外囊泡

John R. Woollard; Amrutesh Puranik; Kyra L. Jordan; Lilach  O. Lerman

doi:10.21769/BioProtoc.3420

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine

使用成像流式细胞术表征从细胞培养基，血浆或尿液分离出的细胞外囊泡

JW John R. Woollard

AP Amrutesh Puranik

KJ Kyra L. Jordan

LL Lilach O. Lerman email

发布: 2019年11月05日第9卷第21期 DOI: 10.21769/BioProtoc.3420 浏览次数: 4248

评审: Xiaoyi ZhengBegona DiazAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Hypertension, featuring study using the protocol.

Nov 2018

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

基于流式的体内方法测定翻译核糖体数量及翻译延长速率

Mina O. Seedhom [...] Jonathan W. Yewdell

2025年01月20日 2088 阅读

使用康可藻红素刺激冷冻保存的猪外周单个核细胞进行增殖检测，并结合FCS ExpressTM 7.18软件分析

Marlene Bravo-Parra [...] Luis G. Giménez-Lirola

2025年06月05日 1285 阅读

利用小鼠脾细胞分离并离体检测CD8⁺ T细胞的分裂与激活

Melissa Dolan [...] John M.L. Ebos

2025年08月20日 1334 阅读

Abstract

The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry method for use with an imaging flow cytometer (Amnis FlowSight) for identifying EV released by specific cell types and excreted into the urine or blood using markers characteristic of particular cells in the kidney. Here we present a protocol utilizing the Amnis FlowSight Imaging Flow Cytometer to identify and quantify EV from the urine of patients with essential hypertension and renovascular disease. Notably, EV isolated from cell culture media and plasma can also be analyzed similarly.

Keywords: Exosomes (外来体)

Extracellular vesicles (细胞外囊泡)

Imaging flow cytometry (成像流式细胞仪)

Biomarkers (生物标计)

Hypertension (高血压)

Renovascular disease (肾血管疾病)

Background

Extracellular vehicles (EVs) are released from cells under normal conditions and their numbers are known to increase when the cells are exposed to stress conditions. Therefore, levels of urinary EVs have been shown to be associated with various kidney disorders such as polycystic kidney disease (Hogan et al., 2009), acute kidney injury (Aghajani Nargesi et al., 2017; Cappuccilli et al., 2018), and various glomerular diseases (Zhang et al., 2019).

We have previously shown that levels of EVs that are increased in patients with hypertension likely originate from podocytes (Kwon et al., 2017) and peritubular capillaries (PTC) (Sun et al., 2018; Zhang et al., 2019). We have also recently shown that urinary levels of EVs reflecting renal cellular senescence are altered in these patients (Santelli et al., 2019). We describe here a more detailed protocol that can be used for the isolation and quantitative characterization of EVs isolated from cell culture media, plasma, and urine (Kwon et al., 2017; Sun et al., 2018; Conley et al., 2019; Zhang et al., 2019). The primary advantages of this method of EV isolation are shorter processing time and obviating the requirement for an ultracentrifuge to isolate the EVs.

Materials and Reagents

Opaque Black 1.5 ml microcentrifuge tubes (Midwest Scientific, catalog number: T7100BK)
Avant clear, 1.5 ml microcentrifuge tubes (Midwest Scientific, catalog number: AVSS1700)
dPBS (Life Technologies, catalog number: 14190-250)
Albumin, bovine serum, fraction V (Sigma, catalog number: A3059)
Invitrogen^TM Total Exosome Isolation reagents
1. From Urine (Thermo Fisher Scientific, Inc., catalog number: 4484452)
2. From Cell culture media (Thermo Fisher Scientific, Inc., catalog number: 4478359)
3. From Plasma (Thermo Fisher Scientific, Inc., catalog number: 4484450)
4. From Serum (Thermo Fisher Scientific, Inc., catalog number: 4478360)
Tag-It Violet^TM proliferation and cell tracking dye (Biolegend, Inc., catalog number: 425101)
PV1/PL-VAP, FITC, anti-human antibody (Lifespan Biosciences, catalog number: LS-C209607-100)
CD31, Brilliant Violet 605, Anti-Human antibody (BioLegend, Inc., catalog number: 303122)
CD144, PE, anti-human antibody (BioLegend, Inc., catalog number: 348506)
Bleach, Clorox (Solution used in the FlowSight during operation)
Isopropanol, HPLC grade (Honeywell, catalog number: AH323-4) (Solution used in the FlowSight during operation)
Coulter Clenz^® (BD Biosciences, catalog number: 8546929) (Solution used in the FlowSight during operation)
Deionized H₂O (Solution used in the FlowSight during operation)
Molecular Probes^TM AbC Total Antibody compensation bead kit (Thermo Fisher Scientific, Inc., catalog number: A10497)
Sucrose (Sigma, BioXtra, catalog number: S7903)
MES: 2-(N-Morpholino) ethanesulfonic acid, 4-Morpholineethanesulfonic acid (Sigma, catalog number: M3671)
DMSO
EV Buffer (pH 7.4) (see Recipes)
5 mM Tag-It Violet^TM (TIV) solution (see Recipes)

Equipment

1 ml pipettor
FlowSight Imaging flow cytometer (Amnis, Inc.) equipped with:
1. 488 nm Laser
2. 405 nm Laser
3. 642 nm laser
4. 785 nm laser (SSC)
5. Quantitative imaging
6. 96 well plate autosampler
Refrigerated Microcentrifuge (Eppendorf, Model 5415R, 48 x 2 ml rotor)
MultiTherm incubated vortexer (Midwest Scientific, catalog number: MTHC-1500)
Barnstead deionized water system (Model)
-80 °C freezer
-20 °C freezer
4 °C refrigerator

Software

Amnis^® IDEAS version 6.2 (Luminex Inc., https://www.luminexcorp.com/imaging-flow-cytometry/), data analysis software
Amnis^® INSPIRE Version 6, data acquisition software
Excel (Microsoft)
JMP version 10.0 (SAS Institute)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Woollard, J. R., Puranik, A., Jordan, K. L. and Lerman, L. O. (2019). Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine. Bio-protocol 9(21): e3420. DOI: 10.21769/BioProtoc.3420.