Publication

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

De Haas, A. H., Boddeke, H. W. G. M., Brouwer, N. & Biber, K., Oct-2007, In : Glia. 55, 13, p. 1374-1384 11 p.

Research output: Contribution to journalArticleAcademicpeer-review

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, immuno-magnetic 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. (c) 2007 Wiley-Liss, Inc.

Original languageEnglish
Pages (from-to)1374-1384
Number of pages11
JournalGlia
Volume55
Issue number13
Publication statusPublished - Oct-2007

    Keywords

  • CNS, microglia, flow cytometry, DRAQ5, chemotaxis, phagocytosis, gene expression, EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS, ADAPTIVE IMMUNE-RESPONSES, MHC CLASS-II, DENDRITIC CELLS, DIFFERENTIAL EXPRESSION, ANTIGEN PRESENTATION, MULTIPLE-SCLEROSIS, INFLAMMATORY CELLS, RESIDENT MICROGLIA, RESTING MICROGLIA

ID: 4605832