Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

Quentin Lemaire; Marie Duhamel; Antonella Raffo-Romero; Michel Salzet; Christophe Lefebvre

doi:10.3791/60118

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

細胞外小胞由来免疫細胞由来の特徴と細胞環境への機能的影響の研究

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10:09 min

June 2, 2020

DOI: 10.3791/60118-v

Quentin Lemaire¹, Marie Duhamel¹, Antonella Raffo-Romero¹, Michel Salzet¹, Christophe Lefebvre¹

¹U1192-Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM),Univ. Lille, INSERM

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Overview

This study details protocols for the isolation of extracellular vesicles (EVs) from microglia and blood macrophages, emphasizing their roles in neurite outgrowth regulation and glioma cell invasion control. The findings aim to enhance our understanding of EV functions as immune mediators within specific microenvironments.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

Extracellular vesicles (EVs) play significant roles in cell communication.
Microglia and macrophages are crucial immune cells in the central nervous system.
Understanding how EVs influence neuronal functions can provide insights into neuroimmune interactions.
Current EV isolation techniques can impact the integrity and activity of the vesicles.

Purpose of Study

To evaluate EVs derived from microglia and blood macrophages in different biological contexts.
To assess the impact of these EVs on neurite outgrowth and glioma cell behavior.
To establish reliable protocols for EV isolation that ensure biological integrity.

Methods Used

The primary method involves cell culture followed by stepwise centrifugation to isolate EVs from conditioned media.
Nanoparticle tracking analysis and Western blot techniques are utilized for EV characterization.
Details include ultra-centrifugation, size exclusion chromatography, and protein extraction methods.
Attention is given to maintaining biological ethics during EV handling.

Main Results

Significantly higher particle counts were observed in specific fractions enriched for EVs.
Western blot analysis confirmed the presence of cardiac stress-related proteins in EV-positive samples.
Electron microscopy identified the size range of isolated EVs, indicating successful isolation methods.

Conclusions

The developed protocols for EV isolation from microglia and macrophages may enhance research on immune-mediated neurodevelopmental processes.
The study provides a framework for future investigations into EV roles in various biological contexts.
Understanding the functional implications of these EVs could inform therapeutic strategies in neurological diseases.

Frequently Asked Questions

What are the advantages of using this EV isolation method?

This protocol ensures higher integrity and activity of isolated EVs compared to traditional techniques, which can lead to more reliable experimental outcomes.

How is the main biological model implemented in the study?

The study utilizes cultured microglia and blood macrophages to derive EVs, allowing exploration of their functional roles in neuronal contexts.

What types of data are obtained from the analysis of isolated EVs?

Data includes particle quantification, protein presence via Western blot, and morphological assessment through electron microscopy, providing insights into EV characteristics.

How can this method be applied or adapted for other studies?

The protocols can be adapted for various cell types and experimental settings to examine the role of EVs in different biological processes and diseases.

What are the key considerations when working with extracellular vesicles?

Maintaining sample integrity during isolation and adhering to biological ethics are crucial for obtaining reliable results from EV research.

本報告書は、ミクログリアまたは血液マクロファージからの細胞外小胞(EV)単離に関する時系列的要件を強調している。ミクログリア由来EVは神経突起の伸長因子として評価され、血液マクロファージ由来EVはインビトロアッセイにおけるC6グリオーマ細胞浸潤の制御で研究された。目標は、これらのEV機能を特定の微小環境における免疫メディエーターとして理解することです。

このプロトコルは、細胞外小胞の良好な分離とEBDFタンパク質の高い数の通知を保証することを可能にします。他の分離手法と比較して、この手法は、より整合性と垂直アクティビティであることを維持します。EVは、LCや病理学的状態でますます研究されています。

この方法は、我々は、EVを特徴付け、研究することを選択した任意のシステムに適用することができます。その内容や生物倫理のためには、培養結果として以下の実験を行うため、EVを反復処理する際には特別な注意が必要である。視覚的なデモンストレーションは、自家製サイズ排除クロマトグラフィーカラムなどの特定の機器をホストし、ナノ粒子カウンターと質量分析計の関与をホストするために重要です。

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