Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Morgan Towriss; Jennifer Kim; Annie Vogel Ciernia

doi:10.3791/65080

JoVE Journal
Neuroscience
Author Produced

This content is Free Access.

JoVE Journal Neuroscience

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

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September 15, 2023

DOI: 10.3791/65080-v

Morgan Towriss¹, Jennifer Kim², Annie Vogel Ciernia¹

¹Department of Biochemistry and Molecular Biology, Djavad Mowafaghian Centre for Brain Health,University of British Columbia, ²Graduate Program in Neuroscience, Djavad Mowafaghian Centre for Brain Health,University of British Columbia

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Overview

This study outlines a protocol for quantifying global histone modifications using intranuclear flow cytometry specifically in isolated brain microglia. The investigation aims to understand the fundamental regulatory mechanisms of gene expression in the brain, particularly how disruptions contribute to neurodevelopmental and neuropsychiatric disorders.

Key Study Components

Area of Science

Neuroscience
Epigenetics
Flow Cytometry

Background

Focus on gene expression regulation in the brain.
Exploration of genetic and environmental risk factors related to brain disorders.
Microglia's role in neurodevelopmental and neuropsychiatric conditions.
Development of a novel high-throughput screening protocol.

Purpose of Study

To provide a method for quantifying histone modifications in microglia.
To facilitate screening of epigenetic modifications before further analysis.
To advance understanding of cellular functions and behavioral impairments related to disorders.

Methods Used

Utilization of intranuclear flow cytometry for quantifying histone modifications.
The biological model involved isolated microglia from mouse brain tissue.
Protocols included immune-enrichment and centrifugation steps for cell isolation.
Detailed steps for preparing tissue samples and analyzing with specific antibodies were provided.
Statistical analysis presented for quantifying histone modification levels via MFI values.

Main Results

Highlighting an increase in histone H3 lysine 27 acetylation due to lipopolysaccharide treatment.
Results indicated normally distributed populations with notable shifts in fluorescence.
Provided quantitative insights into microglia's epigenetic landscape and functional responses.
Demonstrated a fast and efficient method compared to traditional techniques.

Conclusions

This study supports a faster and more quantitative approach to study histone modifications in microglia.
Significant implications for understanding the impact of epigenetic changes on neurodevelopmental and psychiatric disorders.
The method enables high-throughput analysis that could accelerate research in cellular responses and neurobiology.

Frequently Asked Questions

What are the advantages of using intranuclear flow cytometry?

This method is faster and more quantitative than traditional techniques, requiring fewer input cells while allowing multiplexing of analyses.

How is the biological model of brain microglia implemented in this study?

Microglia are isolated from mouse brain tissue and subjected to flow cytometry to analyze histone modifications.

What types of data are obtained using this protocol?

The protocol yields quantitative measures of global histone modifications, providing insights into epigenetic changes in microglia.

Can this method be adapted for other cell types?

While this method is tailored for microglia, it may be adaptable to other immune cell types with similar isolation protocols.

What limitations might this method have?

Potential limitations include the specificity of antibodies used and the need for careful optimization of flow cytometry settings for different experiments.

How can the findings from this protocol impact future research?

The findings can facilitate further studies into the role of epigenetic modifications in neurodevelopmental and psychiatric disorders, guiding therapeutic strategies.

这项工作描述了一种在分离的脑小胶质细胞中使用核内流式细胞术定量全局组蛋白修饰的方案。这项工作还包含用于数据收集的小胶质细胞分离方案。

我们的研究旨在了解控制大脑中基因表达的基本调节机制，我们对了解这些机制的破坏如何导致神经发育和神经精神疾病特别感兴趣。我们专注于了解遗传和环境风险因素如何结合在一起改变细胞功能并产生行为障碍。该协议包括对分离的小胶质细胞中的组蛋白修饰进行高通量筛选的方法，使我们能够在进行高通量测序和功能随访研究之前筛选数十种不同的表观遗传修饰。

我们的协议提供了类似于西方图的全局组织修饰的测量。然而，我们的技术速度更快，需要更少的输入单元，更定量，并且可以进行多路复用。