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

Quantificação de modificações pós-traducionais globais de histona utilizando citometria de fluxo ...

JoVE Journal
Neuroscience
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JoVE Journal Neuroscience

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

Quantificação de modificações pós-traducionais globais de histona utilizando citometria de fluxo intranuclear em microglia cerebral isolada de camundongos

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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.

Este trabalho descreve um protocolo para quantificação de modificações globais de histonas utilizando citometria de fluxo intranuclear em microglia cerebral isolada. O trabalho também contém o protocolo de isolamento da micróglia que foi utilizado para a coleta de dados.

Nossa pesquisa visa entender os mecanismos regulatórios fundamentais que controlam a expressão gênica no cérebro, e estamos particularmente interessados em entender como a interrupção desses mecanismos contribui para condições neurodesenvolvimentais e neuropsiquiátricas. Nosso foco é entender como fatores de risco genéticos e ambientais se combinam para alterar a função celular e produzir prejuízos comportamentais. Este protocolo inclui métodos para triagem de alto rendimento de modificações de histonas em micróglias isoladas, permitindo-nos rastrear dezenas de diferentes modificações epigenéticas antes de nos comprometermos com o sequenciamento de alto rendimento e estudos de acompanhamento funcional.

Nosso protocolo fornece medidas de modificações histológicas globais semelhantes ao gráfico ocidental. No entanto, nossa técnica é mais rápida, requer menos células de entrada, é mais quantitativa e pode ser multiplexada.

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