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

توصيف الحويصلات خارج الخلية المشتقة من الخلايا المناعية ودراسة التأثير الوظيفي على بيئة الخلية

JoVE Journal
Neuroscience

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

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

توصيف الحويصلات خارج الخلية المشتقة من الخلايا المناعية ودراسة التأثير الوظيفي على بيئة الخلية

Full Text

7,329 Views

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

Please note that some of the translations on this page are AI generated. Click here for the English version.

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.

يسلط هذا التقرير الضوء على المتطلبات الزمنية لعزل الحويصلات خارج الخلية (EV) عن الميكروجليا أو الضامة الدموية. تم تقييم المركبات الكهربائية المشتقة من Microglia كجهات تنظيمية للنمو النيوريفي في حين تمت دراسة المركبات الكهربائية المشتقة من الضامة الدم في السيطرة على غزو خلايا الدبقي C6 في الاختبارات المختبرية. والهدف هو فهم أفضل لهذه الوظائف EV كوسطاء المناعة في بيئات دقيقة محددة.

هذا البروتوكول يجعل من الممكن ضمان عزل جيد من الخلايا خارج الخلية وإشعار من عدد أكبر من البروتينات EBDF. مقارنة مع تقنيات العزل الأخرى، هذا واحد يؤكد أنه أكثر سلامة وأنشطة عمودية. يتم دراسة المركبات الكهربائية أكثر فأكثر في LC وكذلك الحالات المرضية.

يمكن تطبيق هذه الطريقة على أي أنظمة نختارها لتوصيف ودراسة المركبات الكهربائية. لهذا المحتوى أو الأخلاقيات البيولوجية مطلوب اهتمام خاص عند التكرار المركبات الكهربائية، كما هو الحال في نتائج الثقافة في التجارب التالية. إن العرض التوضيحي المرئي أمر بالغ الأهمية لاستضافة معدات محددة مثل عمود استبعاد اللوني للحجم محلي الصنع بالإضافة إلى مشاركة عداد الجسيمات النانوية ومطياف الكتلة.

View the full transcript and gain access to thousands of scientific videos