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JoVE Journal
Immunology and Infection
분화 한 인간 단핵구 유래 대식세포에서 세포 외 트랩 방출의 시험관 내 자극 및 시각화
분화 한 인간 단핵구 유래 대식세포에서 세포 외 트랩 방출의 시험관 내 자극 및 시각화
JoVE Journal
Immunology and Infection
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JoVE Journal Immunology and Infection
In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

분화 한 인간 단핵구 유래 대식세포에서 세포 외 트랩 방출의 시험관 내 자극 및 시각화

Full Text
10,677 Views
08:08 min
November 1, 2019

DOI: 10.3791/60541-v

Yunjia Zhang1,2, Benjamin S. Rayner1,2, Mathias Jensen3, Clare L. Hawkins1,2,3

1Heart Research Institute, 2Sydney Medical School,University of Sydney, 3Department of Biomedical Sciences,University of Copenhagen

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

Overview

This protocol outlines a method to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. It also allows for the examination of specific MET protein markers through immunofluorescence staining.

Key Study Components

Area of Science

  • Neuroscience
  • Immunology
  • Cell Biology

Background

  • Extracellular traps are released by immune cells and play a role in innate immunity.
  • Macrophages are critical in the inflammatory response but their MET production is less understood.
  • This protocol uses primary human cells, providing a more physiologically relevant model.
  • No priming step is needed to differentiate monocytes into macrophages, unlike other cell lines.

Purpose of Study

  • To provide a reliable in vitro model for studying MET production in macrophages.
  • To explore the physiological growth of MET structures in vivo.
  • To enhance understanding of macrophage functions in inflammation.

Methods Used

  • Isolation of primary human macrophages from buffy coat preparations.
  • Microscopy for visualizing MET production.
  • Fluorescence staining for detecting MET markers.
  • Immunofluorescence staining for specific protein analysis.

Main Results

  • Successful detection of MET production in live macrophage cultures.
  • Identification of specific MET protein markers through immunofluorescence.
  • Demonstration of the protocol's effectiveness using primary human cells.
  • Insights into the role of macrophages in inflammatory responses.

Conclusions

  • This protocol provides a valuable tool for studying macrophage MET production.
  • It highlights the importance of using primary cells for physiological relevance.
  • The findings may contribute to a better understanding of macrophage functions in health and disease.

Frequently Asked Questions

What are macrophage extracellular traps?
Macrophage extracellular traps (METs) are structures released by macrophages that trap and kill pathogens, playing a role in the immune response.
Why is it important to study METs?
Studying METs helps understand their role in inflammation and immune responses, which can inform therapeutic strategies for inflammatory diseases.
How are primary human macrophages obtained for this protocol?
Primary human macrophages are isolated from buffy coat preparations, which contain a mixture of blood components.
What microscopy techniques are used in this protocol?
The protocol utilizes fluorescence microscopy to visualize MET production in live cell cultures.
Can this protocol be adapted for other cell types?
While this protocol is designed for macrophages, similar techniques may be adapted for other immune cell types.
What is the advantage of using primary cells over cell lines?
Primary cells provide a more accurate representation of in vivo conditions, leading to more relevant biological insights.

여기에 제시된 프로토콜은 현미경 및 형광 염색을 사용하여 살아있는 세포 배양에서 대식세포 외 트랩(MET) 생산을 검출하는 프로토콜이다. 이 프로토콜은 면역형광 염색에 의해 특정 MET 단백질 마커를 검사하기 위해 더 확장될 수 있다.

호중구 및 기타 면역 세포에 의한 세포 외 트랩의 방출은 타고난 면역에서 중요하지만 염증 성 병리학과 강하게 연결됩니다. 이 세포는 선동적인 반응에 있는 중요한 역할을 하기 때문에 아주 작은 대식세포에 있는 이 프로세스에 관하여 알려져 있습니다. 이 프로토콜은 대식세포 세포 외 트랩 또는 마스트 릴리스의 기본 인간 체외 모델을 제공합니다.

그것은 생체 내에서이 구조의 잠재적인 생리적 성장을 연구 하는 우리에 대 한 모델을 제공 합니다. 이 기술의 주요 장점은 이 프로토콜의 세포가 인간 버피 코트 제제로부터 분리된 1차 세포라는 것입니다. 단핵구를 대식세포로 분화하는 데 필요한 프라이밍 단계는 없으며, 이는 THP-1 세포주와 같은 다른 단세포 세포주와 대조된다.

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면역학 및 감염 문제 153 세포외 함정 대식세포 MET 염증 SYTOX 녹색 형광 현미경 검사법

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