Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Michael  G. Shehat; Justine Tigno-Aranjuez

doi:10.3791/59167

Fluxo Cytometric medição da produção de ROS em macrófagos em resposta a FcγR cross-linking

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

Fluxo Cytometric medição da produção de ROS em macrófagos em resposta a FcγR cross-linking

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13:20 min

March 7, 2019

DOI: 10.3791/59167-v

Michael G. Shehat¹, Justine Tigno-Aranjuez¹

¹Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences,University of Central Florida

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Overview

This study demonstrates the reproducible measurement of reactive oxygen species (ROS) in response to Fc纬R crosslinking using flow cytometry. This method is crucial for assessing the role of ROS in various diseases, including chronic granulomatous disease and autoimmune disorders.

Key Study Components

Area of Science

Immunology
Cell Biology
Oxidative Stress

Background

Reactive oxygen species (ROS) measurement is challenging.
Dysregulated ROS production is linked to diseases like chronic granulomatous disease (CGD).
Accurate ROS measurement aids in molecular diagnosis and understanding of immune responses.
Fc纬R activation is significant in studying immunodeficiencies and autoimmune diseases.

Purpose of Study

To demonstrate a reliable method for measuring ROS production.
To assess the impact of Fc纬R crosslinking on phagocyte function.
To explore the implications of ROS in various diseases.

Methods Used

Flow cytometry for ROS detection.
Fluorescent probes to measure ROS levels.
Use of specific antigenic stimuli for accurate assessment.
Reproducibility of the assay for consistent results.

Main Results

Successful measurement of ROS in response to Fc纬R activation.
Demonstrated reproducibility of the flow cytometry method.
Highlighted the role of ROS in immune responses and disease mechanisms.
Provided insights into the relationship between ROS and oxidative stress.

Conclusions

The flow cytometry method is effective for ROS measurement.
Understanding ROS production is vital for diagnosing and treating diseases.
This study contributes to the knowledge of immune function and oxidative stress.

Frequently Asked Questions

What is the significance of measuring ROS?

Measuring ROS is crucial for understanding immune responses and diagnosing diseases like CGD.

How does Fc纬R activation relate to ROS production?

Fc纬R activation triggers ROS production, which is important for phagocyte function and immune defense.

What are the advantages of using flow cytometry for ROS measurement?

Flow cytometry offers reproducibility and the ability to use specific antigenic stimuli for accurate results.

What diseases are associated with dysregulated ROS production?

Dysregulated ROS production is linked to chronic granulomatous disease, autoimmune diseases, and neurodegenerative disorders.

Can this method be used for other types of immune studies?

Yes, this method can be applied to various studies involving immune responses and oxidative stress.

What role does oxidative stress play in diseases?

Oxidative stress contributes to inflammation and cellular damage, playing a role in many diseases.

Este estudo demonstra o uso da citometria de fluxo para detectar a produção de (ROS) de espécies reativas de oxigênio resultantes da ativação do FcγR. Esse método pode ser usado para avaliar as mudanças no antimicrobial e redox função dos fagócitos em resposta aos complexos imunes, opsonized microorganismos ou direto FcγR cross-linking de sinalização.

A medição de espécies reativas de oxigênio ou ROS é notoriamente problemática. Neste vídeo, demonstraremos a medição reprodutível do ROS em resposta ao crosslinking do receptor FC-gama usando sondas fluorescentes e citometria de fluxo. As principais vantagens desta técnica são a reprodutibilidade do ensaio e o uso deste método com estímulos antigênicos específicos, não apenas mitogênios ou indutores ros conhecidos.

A produção de ROS desregulada é central para o desenvolvimento de muitas doenças, como doenças granulomatosas crônicas ou CGD. Medir com precisão a ROS pode constituir uma parte do diagnóstico molecular de CGD. Estudar a produção de ROS mediada por FC é importante no estudo de imunodeficiências, como a CGD, mas também no estudo de doenças autoimunes, ou doenças neurodegenerativas, onde muito ROS leva ao estresse oxidativo e inflamação.

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