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Cited by 81Neuroinflammation and Neurodegeneration MechanismsMultiple Sclerosis Research StudiesNeuroscience and Neuropharmacology Research

Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions

De Haas, Alexander H., Boddeke, Hendricus W. G. M., Brouwer, Nieske, Biber, Knut·2007·Glia
DOI 10.1002/glia.20554
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Abstract

Ex vivo analysis is an accurate and convenient way to study in vivo microglia phenotype and function. However, current microglia isolation protocols for ex vivo analysis show many differences in isolation steps (perfusion, removal of meninges and blood vessels, mechanical dissociation, enzymatic dissociation, density separation, immunomagnetic separation, and fluorescence‐activated cell sorting), often without addressing their effects on microglia purity, number, phenotype, and function. Therefore, the aim of this study was to provide an optimized isolation protocol with emphasis on microglia purity and number to enable ex vivo analysis of adult mouse microglia. The application of this protocol for ex vivo phenotype and functional analysis is corroborated by results from flow cytometry, gene expression analysis, chemotaxis, and phagocytosis assays. In addition, this study shows the possibility to analyze microglia isolated from various central nervous system regions such as optic nerve, striatum, hippocampus, spinal cord, cerebellum, and cerebral cortex. Furthermore, this is the first study presenting DRAQ5 as a superior alternative to propidium iodide for the discrimination between living and dead cells. DRAQ5 staining facilitated the identification of microglia upon flow cytometry without the need of additional fluorescent markers. Along with a favorable emission spectrum, DRAQ5 proved a valuable tool for flow cytometry of microglia. The presented optimized microglia isolation protocol for ex vivo analysis offers the opportunity to obtain more insight into both general and region‐specific microglia behavior. © 2007 Wiley‐Liss, Inc.

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Crossref · 10.1002/glia.20554

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  • Previous reports comparing cell isolation protocols or cells postisolation have often focused on aspects such as cell viability and/or yield over whether the isolated cells are truly reflective of their in vivo state.

    “Previous reports comparing cell isolation protocols or cells postisolation have often focused on aspects such as cell viability and/or yield4,62 over whether the isolated cells are truly reflective of their in vivo state13,14.”

    From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [62]

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Authors
De Haas, A. H., Boddeke, H. W. G. M., Brouwer, N., & Biber, K.
Affiliations
University Medical Center Groningen (NL)
Corresponding author
Biber, Knut
ORCID
Venue
Glia
Publisher
Wiley
Published
2007-07-30
Volume
55
Issue
13
Pages
1374-1384
ISSN
  • 0894-1491 (ISSN-L)
  • 1098-1136
DOI
10.1002/glia.20554
OpenAlex
W2003627032
Cited by
81
References
72
Citations by year
2023: 1 · 2022: 5 · 2021: 4 · 2020: 6 · 2019: 4 · 2018: 1 · 2017: 1 · 2016: 6 · 2015: 7 · 2014: 9 · 2013: 7 · 2012: 3
FWCI
3.80
Type
journal article
Language
en
Open access
closed
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations
  • Glia (publishedVersion)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Multiple Sclerosis Research Studies
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
microglia isolation, DRAQ5, propidium iodide, cell viability staining, microglial phenotype, microglial chemotaxis, microglial phagocytosis, flow cytometry, enzyme dissociation, immunomagnetic separation
MeSH terms
Animals, Anthraquinones, Cell Separation, Central Nervous System, Flow Cytometry, Male, Mice, Inbred C57BL, Perfusion, Staining and Labeling, Microglia, Immunomagnetic Separation, Mice

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