title: Isolation and characterization of primary microglia from post-natal murine brain tissues: a comparison of two methods.
abstract: Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.
authors.affiliation: Department of Pathology, Neuroinflammation Group, Immunology Laboratory, Faculty of Medicine Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia.
authors.forename: Shinsmon
authors.initials: S
authors.lastname: Jose
authors.affiliation: Department of Pathology, Neuroinflammation Group, Immunology Laboratory, Faculty of Medicine Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia.
authors.forename: Shi Wei
authors.initials: SW
authors.lastname: Tan
authors.affiliation: Department of Pathology, Neuroinflammation Group, Immunology Laboratory, Faculty of Medicine Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia.
authors.forename: Chih Kong
authors.initials: CK
authors.lastname: Tong
authors.affiliation: Department of Pathology, Neuroinflammation Group, Immunology Laboratory, Faculty of Medicine Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia.|Genetic Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Sharmili
authors.initials: S
authors.lastname: Vidyadaran
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology* / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000502:physiology*
keywords: CD11b; EasySep; F4/80; microglia; mild trypsinization