Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Yuan Cao; Ji-Yu Qiu; Da Chen; Chen-Yao Li; Shu-Juan Xing; Chen-Xi Zheng; Xin Liu; Yan Jin; Bing-Dong Sui

doi:10.3791/63990

Выделение и анализ прослеживаемых и функционализированных внеклеточных везикул из плазмы и тверды...

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JoVE Journal Biology

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Выделение и анализ прослеживаемых и функционализированных внеклеточных везикул из плазмы и твердых тканей

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09:57 min

October 17, 2022

DOI: 10.3791/63990-v

Yuan Cao*^1,2, Ji-Yu Qiu*^1,3, Da Chen^1,2, Chen-Yao Li^1,4, Shu-Juan Xing^1,5, Chen-Xi Zheng¹, Xin Liu², Yan Jin^1,6, Bing-Dong Sui¹

¹State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology,The Fourth Military Medical University, ²Department of Orthodontics, School of Stomatology,The Fourth Military Medical University, ³Department of VIP Dental Care, School of Stomatology,The Fourth Military Medical University, ⁴School of Basic Medicine,The Fourth Military Medical University, ⁵College of Life Science,Northwest University, ⁶Xi'an Institute of Tissue Engineering and Regenerative Medicine

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Overview

This study presents a protocol for the extraction of extracellular vesicles (EVs) from peripheral blood and solid tissues, allowing for the profiling of surface antigens and protein components. The method enhances the quality and yield of EVs suitable for further functional analysis, particularly in the context of physiological and pathological studies.

Key Study Components

Research Area

Extracellular vesicle biology
Diagnostic applications in cancer and inflammatory diseases
Sample preparation and analysis methods

Background

Importance of extracellular vesicles in cell communication
Need for reliable methods to isolate EVs for analysis
Role of surface markers in disease diagnosis

Methods Used

Isolation of EVs from blood and solid tissues
Flow cytometry for profiling surface antigens
BCA protein assay for quantifying protein content

Main Results

Successfully isolated high-quality EVs with minimal contamination
Identified surface markers relevant to bone and cancer pathology
Demonstrated the feasibility of using this method in diagnostic settings

Conclusions

The study demonstrates a robust method for EV extraction and characterization.
This protocol has significant implications for biological research and clinical diagnostics.

Frequently Asked Questions

What are extracellular vesicles?

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play critical roles in intercellular communication.

How does flow cytometry contribute to the analysis of EVs?

Flow cytometry allows for the detailed profiling of surface antigens on EVs, aiding in their characterization and potential diagnostic use.

What types of tissues can be used for EV extraction?

The protocol is applicable to most blood and solid tissues, enhancing the versatility of EV studies.

Why is sample preparation important for EV analysis?

Proper sample preparation ensures high yield and quality of EVs, minimizing contamination that could affect results.

What role do surface markers play in EVs?

Surface markers are critical for identifying the parent cells of EVs and can provide insights into physiological and pathological conditions.

Is this protocol suitable for clinical applications?

Yes, the protocol is designed to facilitate diagnostic applications for diseases like cancer and inflammatory disorders.

В настоящем протоколе описан способ извлечения внеклеточных везикул из периферической крови и солидных тканей с последующим профилированием поверхностных антигенов и белковых грузов.

Этот протокол обеспечивает реальный способ выделения и сбора правильных внеклеточных везикул из большинства крови и твердых тканей, особенно анализа их источника и содержания белка, что помогает в дальнейших функциональных экспериментах. Помимо высокого выхода и низкой контаминации, основным преимуществом этого протокола является анализ поверхностных антигенов и белковых грузов ЭВ, что полезно для физиологических и патологических исследований. Он облегчает диагностику раковых заболеваний, таких как воспалительные заболевания и остеопороз, путем обнаружения поверхностных маркеров родительских клеток внеклеточных везикул с помощью проточного цитометра.

Количество тканевых внеклеточных везикул имеет решающее значение для следующих экспериментов. Убедитесь, что концентрация внеклеточных везикул не высока. Наберитесь терпения, чтобы выполнить разведение.

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