Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions.
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.
Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Alexander H
AH
de Haas
Hendricus W G M
HW
Boddeke
Nieske
N
Brouwer
Knut
K
Biber
D000818:Animals; D000880:Anthraquinones; D002469:Cell Separation / Q000379:methods*; D002490:Central Nervous System / Q000166:cytology*; D005434:Flow Cytometry; D018189:Immunomagnetic Separation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000378:metabolism / Q000502:physiology; D010477:Perfusion; D013194:Staining and Labeling
C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones