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Open Access 01-12-2017 | Methodology article

Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

Authors: Mark R. Mizee, Suzanne S. M. Miedema, Marlijn van der Poel, Adelia, Karianne G. Schuurman, Miriam E. van Strien, Jeroen Melief, Joost Smolders, Debbie A. Hendrickx, Kirstin M. Heutinck, Jörg Hamann, Inge Huitinga

Published in: Acta Neuropathologica Communications | Issue 1/2017

Abstract

Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.

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Title: Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables
Authors: Mark R. Mizee
Suzanne S. M. Miedema
Marlijn van der Poel
Adelia
Karianne G. Schuurman
Miriam E. van Strien
Jeroen Melief
Joost Smolders
Debbie A. Hendrickx
Kirstin M. Heutinck
Jörg Hamann
Inge Huitinga
Publication date: 01-12-2017
Publisher: BioMed Central
Published in: Acta Neuropathologica Communications / Issue 1/2017
Electronic ISSN: 2051-5960
DOI: https://doi.org/10.1186/s40478-017-0418-8

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Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

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